Apparatus for performing heat-exchanging chemical reactions

ABSTRACT

An apparatus for controlling the temperature of a reaction mixture contained in a chamber of a reaction vessel comprises a thermal surface for contacting a flexible wall of the chamber and an automated machine for increasing the pressure in the chamber. The pressure increase in the chamber is sufficient to force the flexible wall to conform to the thermal surface for good thermal conductance. The apparatus also includes at least one thermal element for heating or cooling the surface to induce a temperature change within the chamber.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. patent application Ser. No.09/468,690, filed Dec. 21, 1999, which is a continuation in part of U.S.application Ser. No. 09/194,374, filed Jul. 25, 2000, now U.S. Pat. No.6,565,815, filed as a national phase stage entry (371) of internationalapplication PCT/US98/03962, filed Mar. 2, 1998. All of theseapplications are incorporated by reference herein for all purposes.

FIELD OF THE INVENTION

The present invention relates to an apparatus for performingheat-exchanging, chemical reactions and for optically detecting areaction product.

BACKGROUND OF THE INVENTION

There are many applications in the field of chemical processing in whichit is desirable to precisely control the temperature of reactionmixtures (e.g., biological samples mixed with chemicals or reagents), toinduce rapid temperature changes in the mixtures, and to detect targetanalytes in the mixtures. Applications for such heat-exchanging chemicalreactions may encompass organic, inorganic, biochemical and molecularreactions, and the like. Examples of thermal chemical reactions includeisothermal nucleic acid amplification, thermal cycling nucleic acidamplification, such as the polymerase chain reaction (PCR), ligase chainreaction (LCR), self-sustained sequence replication, enzyme kineticstudies, homogeneous ligand binding assays, and more complex biochemicalmechanistic studies that require complex temperature changes.Temperature control systems also enable the study of certain physiologicprocesses where a constant and accurate temperature is required.

One of the most popular uses of temperature control systems is for theperformance of PCR to amplify a segment of nucleic acid. In this wellknown methodology, a DNA template is used with a thermostable DNApolymerase, nucleoside triphosphates, and two oligonucleotides withdifferent sequences, complementary to sequences that lie on oppositestrands of the template DNA and which flank the segment of DNA that isto be amplified (“primers”). The reaction components are cycled betweena first temperature (e.g., 95° C.) for denaturing double strandedtemplate DNA, followed by a second temperature (e.g., 40-60° C.) forannealing of primers, and a third temperature (e.g., 70-75° C.) forpolymerization. For some newer assays, the annealing and polymerizationmay be performed at the same temperature (e.g. 55-60° C.), so that onlytwo set point temperatures are required in each thermal cycle. Repeatedcycling provides exponential amplification of the template DNA.

Nucleic acid amplification may be applied to the diagnosis of geneticdisorders; the detection of nucleic acid sequences of pathogenicorganisms in a variety of samples including blood, tissue,environmental, air borne, and the like; the genetic identification of avariety of samples including forensic, agricultural, veterinarian, andthe like; the analysis of mutations in activated oncogenes, detection ofcontaminants in samples such as food; and in many other aspects ofmolecular biology. Polynucleotide amplification assays can be used in awide range of applications such as the generation of specific sequencesof cloned double-stranded DNA for use as probes, the generation ofprobes specific for uncloned genes by selective amplification ofparticular segments of cDNA, the generation of libraries of cDNA fromsmall amounts of mRNA, the generation of large amounts of DNA forsequencing and the analysis of mutations.

A preferred detection technique for chemical or biochemical analysis isoptical interrogation, typically using fluorescence or chemiluminescencemeasurements. For ligand-binding assays, time-resolved fluorescence,fluorescence polarization, or optical absorption is often used. For PCRassays, fluorescence chemistries are often employed.

Conventional instruments for conducting thermal reactions and foroptically detecting the reaction products typically incorporate a blockof metal having as many as ninety-six conical reaction tubes. The metalblock is heated and cooled either by a Peltier heating/cooling apparatusor by a closed-loop liquid heating/cooling system in which liquid flowsthrough channels machined into the block. Such instruments incorporatinga metal block are described in U.S. Pat. No. 5,038,852 to Johnson andU.S. Pat. No. 5,333,675 to Mullis.

These conventional instruments have several disadvantages. First, due tothe large thermal mass of a metal block, the heating and cooling ratesin these instruments are limited to about 1° C./sec resulting in longerprocessing times. For example, in a typical PCR application, fiftycycles may require two or more hours to complete. With these relativelyslow heating and cooling rates, some processes requiring precisetemperature control are inefficient. For example, reactions may occur atthe intermediate temperatures, creating unwanted and interfering sideproducts, such as PCR “primer-dimers” or anomalous amplicons, which aredetrimental to the analytical process. Poor control of temperature alsoresults in over-consumption of expensive reagents necessary for theintended reaction.

A second disadvantage of these conventional instruments is that theytypically do not permit real-time optical detection or continuousoptical monitoring of the chemical reaction. For example, inconventional thermal cycling instruments, optical fluorescence detectionis typically accomplished by guiding an optical fiber to each ofninety-six reaction sites in a metal block. A central high power lasersequentially excites each reaction site and captures the fluorescencesignal through the optical fiber. Since all of the reaction sites aresequentially excited by a single laser and since the fluorescence isdetected by a single spectrometer and photomultiplier tube, simultaneousmonitoring of each reaction site is not possible.

Some of the instrumentation for newer processes requiring faster thermalcycling times has recently become available. One such device isdisclosed by Northrup et al. in U.S. Pat. No. 5,589,136. The deviceincludes a silicon-based, sleeve-type reaction chamber that combinesheaters, such as doped polysilicon for heating, and bulk silicon forconvection cooling. The device optionally includes a secondary tube(e.g., plastic) for holding the sample. In operation, the tubecontaining the sample is inserted into the silicon sleeve. Each sleevealso has its own associated optical excitation source and fluorescencedetector for obtaining real-time optical data. This device permitsfaster heating and cooling rates than the instruments incorporating ametal block described above. There are, however, several disadvantagesto this device in its use of a micromachined silicon sleeve. A firstdisadvantage is that the brittle silicon sleeve may crack and chip. Asecond disadvantage is that it is difficult to micromachine the siliconsleeve with sufficient accuracy and precision to allow the sleeve toprecisely accept a plastic tube that holds the sample. Consequently, theplastic tube may not establish optimal thermal contact with the siliconsleeve.

Another instrument is described by Wittwer et al. in “The LightCycler™.A Microvolume Multisample Fluorimeter with Rapid Temperature Contror”,BioTechniques, Vol. 22, pgs. 176-181, January 1997. The instrumentincludes a circular carousel for holding up to thirty-two samples. Thetemperature of the samples is controlled by a central heating cartridgeand a fan positioned in a central chamber of the carousel. In operation,the samples are placed in capillaries which are held by the carousel,and a stepper motor rotates the carousel to sequentially position eachof the samples over an optics assembly. Each sample is opticallyinterrogated through a capillary tip by epi-illumination. Thisinstrument also permits faster heating and cooling rates than the metalblocks described above. Unfortunately, this instrument is not easilyconfigured for commercial, high throughput diagnostic applications.

SUMMARY

The present invention overcomes the disadvantages of the prior art byproviding an improved apparatus for thermally controlling and opticallyinterrogating a reaction mixture. In contrast to the prior artinstruments described above, the apparatus of the present inventionpermits extremely rapid heating and cooling of the mixture, ensuresoptimal thermal transfer between the mixture and heating or coolingelements, provides real-time optical detection and monitoring ofreaction products with increased detection sensitivity, and is easilyconfigured for automated, high throughput applications. The apparatus isuseful for performing heat-exchanging chemical reactions, such asnucleic acid amplification.

In a preferred embodiment, the apparatus includes a reaction vesselhaving a chamber for holding the mixture. The vessel has a rigid framedefining the side walls of the chamber, and at least one flexible sheetattached to the rigid frame to form a major wall of the chamber. Therigid frame further includes a port and a channel connecting the port tothe chamber to permit easy filling, sealing, and pressurization of thechamber. The apparatus also includes at least one thermal surface forcontacting the flexible major wall of the chamber. The apparatus furtherincludes a device for increasing the pressure in the chamber. Thepressure increase in the chamber is sufficient to force the flexiblemajor wall to contact and conform to the thermal surface, thus ensuringoptimal thermal conductance between the thermal surface and the chamber.The apparatus also includes one or more thermal elements (e.g., aheating element, thermoelectric device, heat sink, fan, or peltierdevice) for heating or cooling the thermal surface to induce atemperature change within the chamber.

In the preferred embodiment, the reaction vessel includes first andsecond flexible sheets attached to opposite sides of the rigid frame toform opposing major walls of the chamber. In this embodiment, theapparatus includes first and second thermal surfaces formed by first andsecond opposing plates positioned to receive the chamber of the vesselbetween. When the pressure in the chamber is increased, the flexiblemajor walls expand outwardly to contact and conform to the innersurfaces of the plates. A resistive heating element, such as a thick orthin film resistor, is coupled to each plate for heating the plates. Inaddition, the apparatus includes a cooling device, such as a fan, forcooling the plates. Each of the plates is preferably constructed of aceramic material and has a thickness less than or equal to 1 mm for lowthermal mass. In particular, it is presently preferred that each of theplates have a thermal mass less than about 5 J/° C., more preferablyless than 3 J/° C., and most preferably less than 1 J/° C. to enableextremely rapid heating and cooling rates.

The apparatus also preferably includes a support structure for holdingthe plates in an opposing relationship to each other. In the preferredembodiment, the support structure comprises a mounting plate having aslot therein, and spacing posts extending from the mounting plate onopposite sides of the slot. Each of the spacing posts has indentationsformed on opposite sides thereof for receiving the edges of the plates.Retention clips hold the edges of the plates in the indentations formedin the spacing posts. The slot in the mounting plate enables insertionof the vessel between the plates.

The pressurization of the chamber ensures that the flexible major wallsof the vessel are forced to conform to the inner surfaces of the plates,thus guaranteeing optimal thermal contact between the major walls andthe plates. In the preferred embodiment, the device for increasingpressure in the chamber comprises a plunger which is inserted into thechannel to compress gas in the vessel and thereby increase pressure inthe chamber. The plunger preferably has a pressure stroke in the channelsufficient to increase pressure in the chamber to at least 2 psi ofabove the ambient pressure external to the vessel, and more preferablyto a pressure in the range of 8 to 15 psi above the ambient pressure. Ina preferred embodiment, the length of the pressure stroke is controlledby one or more pressure control grooves formed in the inner surface ofthe frame that defines the channel. The pressure control grooves extendfrom the port to a predetermined depth in the channel to allow gas toescape from the channel and thereby prevent pressurization of thechamber until the plunger reaches the predetermined depth. When theplunger reaches the predetermined depth, it establishes a seal with thewalls of the channel and begins the pressure stroke. The pressurecontrol grooves provide for highly controllable pressurization of thechamber and help prevent misalignment of the plunger in the channel.

The reaction vessel may be filled and pressurized manually by a humanoperator, or alternatively, the apparatus may include an automatedmachine for filling and pressurizing the vessel. In the automatedembodiment, the apparatus preferably includes a pick-and-place machinehaving a pipette for filling the vessel and having a machine tip forinserting the plunger into the channel after filling. The plungerpreferably includes a cap having a tapered engagement aperture forreceiving and establishing a fit with the machine tip, thereby enablingthe machine tip to pick and place the plunger into the channel.

In another embodiment of the invention, the pressurization of vessel isperformed by a pick-and-place machine having a machine head foraddressing the vessel. The machine head has an axial bore forcommunicating with the channel. The pick-and-place machine also includesa pressure source in fluid communication with the bore for pressurizingthe chamber of the vessel through the bore. In this embodiment, theapparatus also preferably includes a disposable adapter for placing thebore in fluid communication with the channel. The adapter is sized to beinserted into the channel such that the adapter establishes a seal withthe walls of the channel. The disposable adapter preferably includes avalve (e.g., a check valve) for preventing fluid from escaping from thevessel.

In another embodiment of the invention, the device for increasingpressure in the chamber comprises an elastomeric plug which is insertedinto the channel, and a needle inserted through the plug for injectingfluid into the vessel. The needle may be used to inject the reactionmixture into the chamber, followed by air or another suitable gas toincrease pressure in the chamber. The reaction vessel may be filled andpressurized in this manner by a human operator, or alternatively, theapparatus may include an automated machine for filling and pressurizingthe chamber. In the automated embodiment, the apparatus includes amachine for inserting the needle through the plug, and the machineincludes a pressure source for injecting fluid into the vessel throughthe needle.

In another embodiment of the invention, the device for pressurizing thechamber comprises a platen for heat sealing a film or foil to thevessel. The foil is preferably sealed to the portion of the framedefining the port. Heat sealing the film or foil to the vessel seals theport and collapses an end of the channel to reduce the volume of thevessel and thereby increase pressure in the chamber. The reaction vesselmay be heat sealed in this manner by a human operator, or alternatively,the apparatus may include an automated machine, e.g. a press, forsealing the vessel.

The apparatus of the present invention permits real-time monitoring anddetection of reaction products in the vessel with improved opticalsensitivity. In the preferred embodiment, at least two of the side wallsof the chamber are optically transmissive and angularly offset from eachother, preferably by an angle of about 90°. The apparatus furthercomprises an optics system for optically interrogating the mixturecontained in the chamber through the optically transmissive side walls.The optics system includes at least one light source for exciting themixture through a first one of the side walls, and at least one detectorfor detecting light emitted from the chamber through a second one of theside walls.

Optimum optical sensitivity may be attained by maximizing the opticalsampling path length of both the light beams exciting the labeledanalytes in the reaction mixture and the emitted light that is detected.The thin, wide reaction vessel of the present invention optimizesdetection sensitivity by providing maximum optical path length per unitanalyte volume. In particular, the vessel is preferably constructed suchthat the ratio of the width of the chamber to the thickness of thechamber is at least 4:1, and such that the chamber has a thickness inthe range of 0.5 to 2 mm. These parameters are presently preferred toprovide a vessel having a relatively large average optical path lengththrough the chamber, while still keeping the chamber sufficiently thinto allow for extremely rapid heating and cooling of the reactionmixture.

The apparatus of the present invention may be configured as a smallhand-held instrument, or alternatively, as a large instrument withmultiple reaction sites for simultaneously processing hundreds ofsamples. In high throughput embodiments, the plates, heating and coolingelements, and optics are preferably disposed in a single housing to forman independently controllable, heat-exchanging module with detectioncapability. The apparatus includes a base instrument for receiving aplurality of such modules, and the base instrument includes processingelectronics for independently controlling the operation of each module.Each module provides a reaction site for thermally processing a samplecontained in a reaction vessel and for detecting one or more targetanalytes in the sample. The apparatus may also include a computer forcontrolling the base instrument.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a partially exploded, isometric view of a reaction vesselaccording to a first embodiment of the present invention in which themajor walls of the reaction chamber are removed to show the interior ofthe chamber.

FIG. 2 is a front view of the vessel of FIG. 1.

FIG. 3 is a top view of a plunger cap of the vessel of FIG. 1.

FIG. 4 is another front view of the vessel of FIG. 1.

FIG. 5 is a side view of the vessel of FIG. 1 inserted into a thermalsleeve formed by opposing plates.

FIG. 6 is a front view of one of the plates of FIG. 5.

FIGS. 7A-7D are schematic, cross-sectional views of a plunger beinginserted into a channel of the reaction vessel of FIG. 1.

FIG. 8 is a schematic, front view of a heat-exchanging module accordingto the present invention having a thermal sleeve, a pair of opticsassemblies, and a cooling system. The reaction vessel of FIG. 1 isinserted into the thermal sleeve.

FIG. 9 is an exploded view of a support structure for holding the platesof FIG. 5.

FIGS. 10-11 are assembled views of the support structure of FIG. 9.

FIG. 12 is an isometric view of the reaction vessel of FIG. 1 insertedbetween the plates of FIG. 5.

FIG. 13 is an isometric view showing the exterior of one the opticsassemblies of FIG. 8.

FIG. 14 is an isometric view of the optics assembly of FIG. 13, theplates of FIG. 5 in contact with the optics assembly, and the vessel ofFIG. 1 positioned above the plates.

FIGS. 15A and 15B are graphs showing the excitation and emissionspectra, respectively, of four dyes often used in thermal reactions.

FIG. 15C shows the effects of filtering the outputs of green and blueLEDs to provide distinct excitation wavelength ranges.

FIG. 15D shows the effects of filtering light emitted from each of thefour dyes of FIGS. 15A-B to form distinct emission wavelength ranges.

FIG. 16 is a plan view of an optical excitation assembly of the moduleof FIG. 8.

FIG. 17 is an exploded view of the excitation assembly of FIG. 16.

FIG. 18 is a plan view of an optical detection assembly of the module ofFIG. 8.

FIG. 19 is an exploded view of the detection assembly of FIG. 18.

FIG. 20 is an isometric view of a multi-site reactor system according tothe present invention.

FIG. 21 is a schematic, block diagram of another multi-site reactorsystem having multiple thermal cycling instruments daisy-chained to acomputer and a power source.

FIG. 22 is a schematic, block diagram of a base instrument of the systemof FIG. 20.

FIG. 23 is a schematic, block diagram of the electronic components ofthe module of FIG. 8.

FIG. 24 is a schematic diagram of a pick-and-place machine having apipette for filling the reaction vessel of FIG. 1.

FIG. 25 is a schematic diagram of the pick-and-place machine of FIG. 24inserting a plunger into the vessel of FIG. 1.

FIG. 26 is a schematic, front view of a reaction vessel according toanother embodiment of the invention.

FIG. 27 is a schematic, cross sectional view of a reaction vesselaccording to an alternative embodiment of the invention.

FIG. 28 is a partially exploded, isometric view of a reaction vesselaccording to another embodiment of the invention in which the majorwalls of the reaction chamber are removed to show the interior of thechamber.

FIG. 29 is a schematic front view of the vessel of FIG. 28.

FIG. 30 is a top view of a plunger cap of the vessel of FIG. 28.

FIG. 31 is a schematic front view of a reaction vessel according toanother embodiment of the invention.

FIG. 32 is a top view of a plunger cap of the vessel of FIG. 31.

FIG. 33 is a schematic diagram of a pick-and-place machine forpressurizing a reaction vessel according to another embodiment of theinvention.

FIGS. 34-35 are a schematic diagrams of a pick-and-place machine using adouble-bore needle to fill and pressurize a reaction vessel according toan alternative embodiment of the invention.

FIGS. 36-37 are schematic diagrams of a pick-and-place machine using asingle-bore needle to fill and pressurize a reaction vessel according toanother embodiment of the invention.

FIGS. 38-39 are schematic diagrams of a press machine having a platenfor sealing a port of a reaction vessel according to an alternativeembodiment of the invention.

DETAILED DESCRIPTION

The present invention provides an apparatus for thermally controllingand optically interrogating a reaction mixture, e.g., a sample mixedwith one or more chemicals or reagents. The sample may also be mixedwith diluents or buffers. The sample may be an aqueous solutioncontaining particles, cells, microorganisms, ions, or small and largemolecules, such as proteins and nucleic acids, etc. In a particular use,the sample may be a bodily fluid (e.g., blood, urine, saliva, sputum,seminal fluid, spinal fluid, mucus, or other bodily fluids).Alternatively, the sample may be a solid made soluble in a liquid or thesample may be an environmental sample such as ground or waste water,soil extracts, pesticide residues, or airborne spores placed in aliquid.

In a preferred embodiment, the apparatus includes a reaction vessel forholding the mixture and a heat-exchanging module into which the vesselis inserted for thermal processing and optical detection. Theheat-exchanging module includes a pair of opposing plates between whichthe vessel is inserted for thermal processing, one or more heating orcooling elements for heating or cooling the plates, and optics foroptically interrogating the reaction mixture contained in the vessel.The apparatus also includes a base unit with processing electronics forreceiving a plurality of such heat-exchanging modules and forindependently controlling each module. The apparatus may also include acontroller, such as a personal computer or network computer, thatprovides a user interface to the apparatus and controls the operation ofthe base unit. The apparatus is useful for performing heat-exchangingchemical reactions, such as nucleic acid amplification, and foroptically detecting target analytes.

FIGS. 1-25 illustrate a preferred embodiment of the invention. FIG. 1shows a partially exploded view of a reaction vessel 12 according to thepreferred embodiment, and FIG. 2 shows a front view of the vessel 12.The vessel 12 includes a reaction chamber 17 for holding a reactionmixture for thermal processing and optical interrogation. The vessel 12is designed for optimal heat transfer to and from the mixture and forefficient optical viewing of the mixture. The thin shape of the vesselcontributes to optimal thermal kinetics by providing large surfaces forthermal conduction. In addition, the side walls of the vessel 12 provideoptical windows into the chamber 17 so that the entire reaction mixturecan be optically interrogated in real-time as the chemical reactionoccurs.

In more detail to FIGS. 1-2, the reaction vessel 12 includes a rigidframe 16 that defines the side walls 19A, 19B, 20A, 20B of the reactionchamber 17. The rigid frame 16 also includes a port 14 and a channel 28that connects the port 14 to the chamber 17. The vessel also includesthin, flexible sheets attached to opposite sides of the rigid frame 16to form opposing major walls 18 of the chamber. (The major walls 18 areshown in FIG. 1 exploded from the rigid frame 16 for illustrativeclarity). The reaction chamber 17 is thus defined by the rigid sidewalls 19A, 19B, 20A, 20B of the frame 16 and by the flexible major walls18 which are sealed to opposite sides of the frame.

The major walls 18 facilitate optimal thermal conductance to thereaction mixture contained in the chamber 17. Each of the walls 18 issufficiently flexible to contact and conform to a respective thermalsurface, thus providing for optimal thermal contact and heat transferbetween the thermal surface and the reaction mixture contained in thechamber 17. Furthermore, the flexible walls 18 continue to conform tothe thermal surfaces if the shape of the surfaces changes due to thermalexpansion or contraction during the course of the heat-exchangingoperation.

As shown in FIG. 5, the thermal surfaces for contacting the flexiblewalls 18 are preferably formed by a pair of opposing plates 50A, 50Bpositioned to receive the chamber 17 between them. When the chamber 17of the vessel is inserted between the plates 50A, 50B, the innersurfaces of the plates contact the walls 18 and the flexible wallsconform to the surfaces of the plates. The plates are preferably spaceda distance from each other equal to the thickness T of the chamber 17 asdefined by the thickness of the frame 16. In this position, minimal orno gaps are found between the plate surfaces and the walls 18. Theplates may be heated and cooled by various thermal elements to inducetemperature changes within the chamber 17, as is described in greaterdetail below.

The walls 18 are preferably flexible films of polymeric material such aspolypropylene, polyethylene, polyester, or other polymers. The films mayeither be layered, e.g., laminates, or the films may be homogeneous.Layered films are preferred because they generally have better strengthand structural integrity than homogeneous films. In particular, layeredpolypropylene films are presently preferred because polypropylene is notinhibitory to PCR. Alternatively, the walls 18 may comprise any othermaterial that may be formed into a thin, flexible sheet and that permitsrapid heat transfer. For good thermal conductance, the thickness of eachwall 18 is preferably between about 0.003 to 0.5 mm, more preferablybetween 0.01 to 0.15 mm, and most preferably between 0.025 to 0.08 mm.

Referring again to FIGS. 1-2, the reaction vessel 12 also includes aplunger 22 that is inserted into the channel 28 after filling thechamber 17 with the reaction mixture. The plunger 22 compresses gas inthe vessel 12 thereby increasing pressure in the chamber 17 andoutwardly expanding the flexible walls 18. The gas compressed by theplunger 22 is typically air filling the channel 28. The pressurizationof the chamber 17 is important because it forces the walls 18 againstthe surfaces of the plates 50A, 50B (see FIG. 5) and ensures that thewalls 18 fully contact and conform to the inner surfaces of the plates,thus guaranteeing optimal thermal conductance between the plates 50A,50B and the chamber 17.

Referring again to FIGS. 1-2, the plunger may comprise any devicecapable of establishing a seal with the walls of the channel 28 and ofcompressing gas in the vessel. Such devices include, but are not limitedto, pistons, plugs, or stoppers. The plunger 22 of the preferredembodiment includes a stem 30 and a piston 32 on the stem. When theplunger 22 is inserted into the channel 28, the piston 32 establishes aseal with the inner walls of the channel and compresses air in thechannel. The piston 32 is preferably a cup integrally formed (e.g.,molded) with the stem 30. Alternatively, the piston 32 may be a separateelastomeric piece attached to the stem.

The plunger 22 also preferably includes an alignment ring 34 encirclingthe stem for maintaining the plunger 22 in coaxial alignment with thechannel 28 as the plunger is inserted into the channel. The alignmentring 34 is preferably integrally formed (e.g., molded) with the stem 30.The stem 30 may optionally includes support ribs 44 for stiffening andstrengthening the stem. The plunger 22 also includes a plunger cap 36attached to the stem 30. As shown in FIG. 2, the cap 36 includes a snapring 38 and the vessel includes an annular recess 23 encircling the port14 for receiving the snap ring 38.

The cap 36 may optionally include a lever portion 40 which is lifted toremove the plunger 22 from the channel 28.

Referring to FIG. 7A, the rigid frame 16 has an inner surface 41defining the channel 28. The inner surface 41 preferably has one or morepressure control grooves 42 formed therein. In the preferred embodiment,the inner surface has four pressure control grooves (only three shown inthe view of FIG. 7A) spaced equidistantly about the circumference of thechannel 28. The pressure control grooves 42 extend from the port 14 to apredetermined depth D1 in the channel 28. The pressure control grooves42 allow gas to escape from the channel 28 and thus preventpressurization of the chamber 17 until the piston 32 reaches the depthD1 in the channel. When the piston 32 reaches the depth D1, the pistonestablishes an annular seal with the walls of the channel 28 and beginsto compress air trapped in the channel. The compression of the trappedair causes the desired pressurization of the chamber 17.

The stroke of the plunger 22 into the channel 28 is fully illustrated inFIGS. 7A-7D. As shown in FIG. 7A, prior to inserting the plunger 22 intothe channel 28, the chamber 17 is filled with the desired reactionmixture R. Specific methods for filling the chamber (e.g., pipetting)are discussed in detail below. The reaction mixture R fills the vessel12 to a liquid surface level S. Also prior to inserting the plunger 22into the channel 28, the channel 28 contains air having pressure equalto the pressure of the atmosphere external to the vessel, hereinaftercalled ambient pressure. The ambient pressure is usually standardatmospheric pressure, e.g., about 14.7 pounds per square inch (psi). Asshown in FIG. 7B, when the plunger 22 is first inserted into the channel28, the piston 32 begins to displace the air in the channel. Thedisplaced air escapes from the channel 28 through the pressure controlgrooves 42.

Referring now to FIG. 7C, when the piston 32 reaches the depth D1 atwhich the pressure control grooves end, the piston 32 establishes anannular seal with the walls of the channel 28 and begins to compress airtrapped in the channel between the piston 32 and the surface level S ofthe reaction mixture. The reaction mixture is usually a liquid andtherefore substantially incompressible by the piston. The air trapped inthe channel 28, however, may be compressed to increase pressure in thechamber. As shown in FIG. 7D, as the plunger 22 is inserted further intothe channel 28, the alignment ring 34 keeps the plunger 22 coaxiallyaligned with the channel 28 as the piston 32 continues to compress airtrapped in the channel. When the plunger 22 is fully inserted in thechannel 28, the snap ring 38 snaps into the annular recess 23, endingthe plunger stroke.

When the plunger 22 is fully inserted, the piston 32 seals the channel28 at a depth D2 which is lower than the depth D1 at which the pressurecontrol grooves 42 terminate. The distance D3 traveled by the piston 32between depths D1 and D2, i.e. the distance of the pressure stroke,determines the amount of pressurization of the chamber 17. Referringagain to FIG. 5, the pressure in the chamber 17 should be sufficientlyhigh to ensure that the flexible major walls 18 of the chamber outwardlyexpand to contact and conform to the surfaces of the plates 50A, 50B.The pressure should not be so great, however, that the flexible walls 18burst, become unattached from the rigid frame 16, or deform the frame orplates.

It is presently preferred to pressurize the chamber to a pressure in therange of 2 to 50 psi above ambient pressure. This range is presentlypreferred because 2 psi is generally enough pressure to ensureconformity between the flexible walls 18 and the surfaces of the plates50A, 50B, while pressures above 50 psi may cause bursting of the walls18 or deformation of the frame 16 or plates 50A, 50B. More preferably,the chamber 17 is pressurized to a pressure in the range of 8 to 15 psiabove ambient pressure. This range is more preferred because it issafely within the practical limits described above, i.e. pressures of 8to 15 psi are usually more than enough to ensure that the flexible walls18 contact and conform to the surfaces of the plates 50A, SOB, but aresignificantly lower than the pressures that might burst the walls 18 ordeform the frame 16.

Referring again to FIG. 7D, the desired pressurization of the chamber 17may be achieved by proper design of the plunger 22, channel 28, andpressure control grooves 42 and by use of the equation:P ₁ *V ₁ =P ₂ *V ₂;where:P₁ is equal to the pressure in the vessel 12 prior to insertion of theplunger 22;V₁ is equal to the volume of the channel 28 between the liquid surfacelevel S and the depth D₁ to which the pressure control grooves 42extend;P₂ is equal to the desired final pressure in the chamber 17 afterinsertion of the plunger 22 into the channel 28; andV₂ is equal to the volume of the channel 28 between the liquid surfacelevel S and the depth D₂ at which the piston 32 establishes a seal withthe walls of the channel 28 when the plunger 22 is fully inserted intothe channel.

To ensure the desired pressurization P₂ of the chamber 17, one shouldsize the channel 28 and pressure stroke distance D₃ such that the ratioof the volumes V₁:V₂ is equal to the ratio of the pressures P₂:P₁. Anengineer having ordinary skill in the art will be able to selectsuitable values for the volumes V₁ and V₂ using the description andequation given above. For example, in the presently preferredembodiment, the initial pressure P₁ in the vessel is equal to standardatmospheric pressure of about 14.7 psi, the volume V₁ is equal to 110μl, the depth D₁ is equal to 0.2 inches, the depth D₂ is equal to 0.28inches to give a pressure stroke distance D₃ of 0.08 inches, and thevolume V₂ is equal to 60 μl to give a final pressure P₂ of about 26.7psi (the desired 12 psi above ambient pressure). This is just oneexample of suitable dimensions for the vessel 12 and is not intended tolimit the scope of the invention. Many other suitable values may beselected.

In selecting suitable dimensions for the channel 28 and pressure strokedistance D₃ (and thus the volumes V₁, V₂), there is no theoretical limitto how large or small the dimensions may be. It is only important thatthe ratio of the volumes V1:V₂ yield the desired final desired pressureP₂ in the chamber. As a practical matter, however, it is presentlypreferred to design the vessel such that the distance D₃ of the pressurestroke is at least 0.05 inches, i.e., so that the plunger 22 when fullyinserted into the channel 28 extends to a depth D₂ that is at least 0.05inches below the depth D₁ at which the pressure control grooves end.This minimum length of the pressure stroke is preferred to reduce ormake negligible the effect that any manufacturing or operating errorsmay have on the pressurization of the chamber. For example, the lengthof the pressure stroke may differ slightly from vessel to vessel due tomanufacturing deviations, or the volume of air compressed may vary dueto operator error in filling the vessel (e.g., different fill levels).If the vessel is designed to have a sufficiently long pressure stroke,however, such variances will have a lesser or negligible effect on theratio of volumes V₁:V₂ and suitable pressurization of the chamber willstill occur. In addition, to provide a safety margin for manufacturingor operator errors, one should select a pressure stroke sufficient toachieve a final pressure P₂ that is safely higher (e.g., at least 3 psihigher) than the minimum pressure needed to force the flexible walls ofthe chamber against the inner surfaces of the plates. With such a safetymargin, any deviations in the final pressure due to manufacturingdeviations or errors in filling the chamber will have a negligibleeffect and suitable pressurization of the chamber 17 will still occur.As stated above, the plunger stroke is preferably designed to increasepressure in the chamber 17 to a pressure in the range of 8 to 15 psiabove ambient pressure to provide the safety margin.

The pressure control grooves 42 provide several important advantages.First, the pressure control grooves 42 provide a simple mechanism forprecisely and accurately controlling the pressure stroke of the plunger22, and hence the pressurization of the chamber 17. Second, the pressurecontrol grooves 42 allow the plunger 22 to become fully aligned with thechannel 28 before the pressure stroke begins and thus prevent theplunger from becoming misaligned or cocked in the channel. This ensuresa highly consistent pressure stroke. Although it is possible for thevessel to have only one pressure control groove, it is preferable forthe vessel to have multiple pressure control grooves (e.g., 2 to 6grooves) spaced equidistantly about the circumference of the channel 28.Referring again to FIG. 7A, the pressure control grooves 42 preferablycut about 0.01 to 0.03 inches into the surface 41 defining the channel28. This range is preferred so that the pressure control grooves 42 arelarge enough to allow air to escape from the channel 28, but do not cutso deeply into the surface 41 that they degrade the structural integrityof the frame 16.

Although the pressure control grooves 42 are highly preferred, it isalso possible to construct the vessel 12 without the pressure controlgrooves and still achieve the desired pressurization of the chamber 17.One disadvantage of this embodiment is that the plunger 22 may becomemisaligned or cocked in the channel 28 during the pressure stroke sothat less consistent results are achieved. In embodiments in which thevessel lacks pressure control grooves, the pressure stroke of theplunger 22 begins when the piston 32 enters the channel 28 andestablishes a seal with the walls of the channel. In these embodiments,the volume V₁ (for use in the equation above) is equal to the volume ofthe channel 28 between the liquid surface level S and the port 14 wherethe piston 32 first establishes a seal with the walls of the channel. Toensure the desired pressurization P₂ of the chamber 17, one should sizethe channel 28 and length of the pressure stroke such that the ratio ofthe volumes V₁:V₂ is equal to the ratio of the pressures P₂:P₁. Asdescribed previously, the minimum length of the pressure stroke ispreferably 0.05 inches to minimize the effect of any manufacturing oroperational deviations.

Referring again to FIG. 2, the vessel 12 also preferably includesoptical windows for in situ optical interrogation of the reactionmixture in the chamber 17. In the preferred embodiment, the opticalwindows are the side walls 19A, 19B of the rigid frame 16. The sidewalls 19A, 19B are optically transmissive to permit excitation of thereaction mixture in the chamber 17 through the side wall 19A anddetection of light emitted from the chamber 17 through the side wall19B. Arrows A represent illumination beams entering the chamber 17through the side wall 19A and arrows B represent emitted light (e.g.,fluorescent emission from labeled analytes in the reaction mixture)exiting the chamber 17 through the side wall 19B.

The side walls 19A, 19B are preferably angularly offset from each other.It is usually preferred that the walls 19A, 19B are offset from eachother by an angle of about 90°. A 90° angle between excitation anddetection paths assures that a minimum amount of excitation radiationentering through the wall 19A will exit through wall 19B. In addition,the 90° angle permits a maximum amount of emitted light, e.g.fluorescence, to be collected through wall 19B. The walls 19A, 19B arepreferably joined to each other to form a “V” shaped intersection at thebottom of the chamber 17. Alternatively, the angled walls 19A, 19B neednot be directly joined to each other, but may be separated by anintermediary portion, such as another wall or various mechanical orfluidic features which do not interfere with the thermal and opticalperformance of the vessel. For example, the walls 19A, 19B may meet at aport which leads to another processing area in communication with thechamber 17, such as an integrated capillary electrophoresis area. In thepresently preferred embodiment, a locating tab 27 extends from the frame16 below the intersection of walls 19A, 19B. The locating tab 27 is usedto properly position the vessel 12 in a heat-exchanging module describedbelow with reference to FIG. 8.

Optimum optical sensitivity may be attained by maximizing the opticalpath length of the light beams exciting the labeled analytes in thereaction mixture and the emitted light that is detected, as representedby the equation:I _(o) /I _(i) =C*L*A,where I_(o) is the illumination output of the emitted light in volts,photons or the like, C is the concentration of analyte to be detected,I_(i) is the input illumination, L is the path length, and A is theintrinsic absorptivity of the dye used to label the analyte.

The thin, flat reaction vessel 12 of the present invention optimizesdetection sensitivity by providing maximum optical path length per unitanalyte volume. Referring to FIGS. 4-5, the vessel 12 is preferablyconstructed such that each of the sides walls 19A, 19B, 20A, 20B of thechamber 17 has a length L in the range of 1 to 15 mm, the chamber has awidth W in the range of 1.4 to 20 mm, the chamber has a thickness T inthe range of 0.5 to 5 mm, and the ratio of the width W of the chamber tothe thickness T of the chamber is at least 2:1. These parameters arepresently preferred to provide a vessel having a relatively largeaverage optical path length through the chamber, i.e. 1 to 15 mm onaverage, while still keeping the chamber sufficiently thin to allow forextremely rapid heating and cooling of the reaction mixture containedtherein. The average optical path length of the chamber 17 is thedistance from the center of the side wall 19A to the center of thechamber 17 plus the distance from the center of the chamber 17 to thecenter of the side wall 19B. As used herein, the thickness T of thechamber 17 is defined as the thickness of the chamber prior to theoutward expansion of the major walls, i.e. the thickness T of thechamber is defined by the thickness of the frame 16.

More preferably, the vessel 12 is constructed such that each of thesides walls 19A, 19B, 20A, 20B of the chamber 17 has a length L in therange of 5 to 12 mm, the chamber has a width W in the range of 7 to 17mm, the chamber has a thickness T in the range of 0.5 to 2 mm, and theratio of the width W of the chamber to the thickness T of the chamber isat least 4:1. These ranges are more preferable because they provide avessel having both a larger average optical path length (i.e., 5 to 12mm) and a volume capacity in the range of 12 to 100 μl while stillmaintaining a chamber sufficiently thin to permit extremely rapidheating and cooling of a reaction mixture. The relatively large volumecapacity provides for increased sensitivity in the detection of lowconcentration analytes, such as nucleic acids.

In the preferred embodiment, the reaction vessel 12 has a diamond-shapedchamber 17 defined by the side walls 19A, 19B, 20A, 20B, each of theside walls has a length of about 10 mm, the chamber has a width of about14 mm, the chamber has a thickness T of 1 mm as defined by the thicknessof the frame 16, and the chamber has a volume capacity of about 100 μl.This reaction vessel provides a relatively large average optical pathlength of 10 mm through the chamber 17. Additionally, the thin chamberallows for extremely rapid heating and/or cooling of the reactionmixture contained therein. The diamond-shape of the chamber 17 helpsprevent air bubbles from forming in the chamber as it is filled with thereaction mixture and also aids in optical interrogation of the mixture.

The frame 16 is preferably made of an optically transmissive material,e.g., a polycarbonate or clarified polypropylene, so that the side walls19A, 19B are optically transmissive. As used herein, the term opticallytransmissive means that one or more wavelengths of light may betransmitted through the walls. In the preferred embodiment, theoptically transmissive walls 19A, 19B are substantially transparent. Inaddition, one or more optical elements may be present on the opticallytransmissive side walls 19A, 19B. The optical elements may be designed,for example, to maximize the total volume of solution which isilluminated by a light source, to focus excitation light on a specificregion of the chamber 17, or to collect as much fluorescence signal fromas large a fraction of the chamber volume as possible. In alternativeembodiments, the optical elements may comprise gratings for selectingspecific wavelengths, filters for allowing only certain wavelengths topass, or colored lenses to provide filtering functions. The wallsurfaces may be coated or comprise materials such as liquid crystal foraugmenting the absorption of certain wavelengths. In the presentlypreferred embodiment, the optically transmissive walls 19A, 19B aresubstantially clear, flat windows having a thickness of about 1 mm.

As shown in FIG. 2, the side walls 20A, 20B preferably includesreflective faces 21 which internally reflect light trying to exit thechamber 17 through the side walls 20A, 20B. The reflective faces 21 arearranged such that adjacent faces are angularly offset from each otherby about 90°. In addition, the frame 16 defines open spaces between theside walls 20A, 20B and support ribs 15. The open spaces are occupied byambient air that has a different refractive index than the materialcomposing the frame (e.g., plastic). Due to the difference in therefractive indexes, the reflective faces 21 are effective for internallyreflecting light trying to exit the chamber through the walls 20A, 20Band provide for increased detection of optical signal through the walls19A, 19B. In the preferred embodiment, the optically transmissive sidewalls 19A, 19B define the bottom portion of the diamond-shaped chamber17, and the retro-reflective side walls 20A, 20B define the top portionof the chamber.

The reaction vessel 12 may be used in manual operations performed byhuman technicians or in automated operations performed by machines, e.g.pick-and-place machines. As shown in FIG. 1, for the manual embodiments,the vessel 12 preferably includes finger grips 26 and a leash 24 thatconveniently attaches the plunger 22 to the body of the vessel 12. Asshown in FIG. 3, for automated embodiments, the plunger cap 36preferably includes a tapered engagement aperture 46 for receiving andestablishing a fit with a robotic arm or machine tip (not shown in FIG.3), thus enabling the machine tip to pick and place the plunger in thechannel. The engagement aperture 46 preferably has tapered side wallsfor establishing a friction fit with the machine tip. Alternatively, theengagement aperture may be designed to establish a vacuum fit with themachine tip. The plunger cap 36 may optionally include alignmentapertures 48A, 48B used by the machine tip to properly align the plungercap 36 as the plunger is inserted into the channel, as is described ingreater detail below with reference to FIG. 25.

A preferred method for fabricating the reaction vessel 12 will now bedescribed with reference to FIGS. 1-2. The reaction vessel 12 may befabricated by first molding the rigid frame 16 using known injectionmolding techniques. The frame 16 is preferably molded as a single pieceof polymeric material, e.g., clarified polypropylene. After the frame 16is produced, thin, flexible sheets are cut to size and sealed toopposite sides of the frame 16 to form the major walls 18 of the chamber17.

The major walls 18 are preferably cast or extruded films of polymericmaterial, e.g., polypropylene films, that are cut to size and attachedto the frame 16 using the following procedure. A first piece of film isplaced over one side of the bottom portion of the frame 16. The frame 16preferably includes a tack bar 47 for aligning the top edge of the film.The film is placed over the bottom portion of the frame 16 such that thetop edge of the film is aligned with the tack bar 47 and such that thefilm completely covers the bottom portion of the frame 16 below the tackbar 47. The film should be larger than the bottom portion of the frame16 so that it may be easily held and stretched flat across the frame.The film is then cut to size to match the outline of the frame byclamping to the frame the portion of the film that covers the frame andcutting away the portions of the film that extend past the perimeter ofthe frame using, e.g., a laser or die. The film is then tack welded tothe frame, preferably using a laser.

The film is then sealed to the frame 16, preferably by heat sealing.Heat sealing is presently preferred because it produces a strong sealwithout introducing potential contaminants to the vessel as the use ofadhesive or solvent bonding techniques might do. Heat sealing is alsosimple and inexpensive. At a minimum, the film should be completelysealed to the surfaces of the side walls 19A, 19B, 20A, 20B. Morepreferably, the film is additionally sealed to the surfaces of thesupport ribs 15 and tack bar 47. The heat sealing may be performedusing, e.g., a heated platen. An identical procedure may be used to cutand seal a second sheet to the opposite side of the frame 16 to completethe chamber 17.

Many variations to this fabrication procedure are possible. For example,in an alternative embodiment, the film is stretched across the bottomportion of the frame 16 and then sealed to the frame prior to cuttingthe film to size. After sealing the film to the frame, the portions ofthe film that extend past the perimeter of the frame are cut away using,e.g., a laser or die.

The plunger 22 is also preferably molded from polymeric material,preferably polypropylene, using known injection molding techniques. Asshown in FIG. 1, the frame 16, plunger 22, and leash 24 connecting theplunger to the frame may all be formed in the same mold to form aone-piece part. This embodiment of the vessel is especially suitable formanual use in which a human operator fills the vessel and inserts theplunger 22 into the channel 28. The leash 24 ensures that the plunger 22is not lost or dropped on the floor. Alternatively, as shown in FIG. 2,the plunger 22 may be molded separately from the frame 16 so that theplunger and frame are separate pieces. This embodiment is especiallysuitable for automated use of the vessel in which the plunger 22 ispicked and placed into the channel 28 by an automated machine.

Although it is presently preferred to mold the frame 16 as a singlepiece, it is also possible to fabricate the frame from multiple pieces.For example, the side walls 19A, 19B forming the angled optical windowsmay be molded from polycarbonate, which has good optical transparency,while the rest of the frame is molded from polypropylene, which isinexpensive and compatible with PCR. The separate pieces can be attachedtogether in a secondary step. For example, the side walls 19A, 19B maybe press-fitted and/or bonded to the remaining portion of the frame 16.The flexible walls 18 may then be attached to opposite sides of theframe 16 as previously described.

Referring again to FIG. 5, the plates 50A, 50B may be made of variousthermally conductive materials including ceramics or metals. Suitableceramic materials include aluminum nitride, aluminum oxide, berylliumoxide, and silicon nitride. Other materials from which the plates may bemade include, e.g., gallium arsenide, silicon, silicon nitride, silicondioxide, quartz, glass, diamond, polyacrylics, polyamides,polycarbonates, polyesters, polyimides, vinyl polymers, and halogenatedvinyl polymers, such as polytetrafluoroethylenes. Other possible platematerials include chrome/aluminum, superalloys, zircaloy, aluminum,steel, gold, silver, copper, tungsten, molybdenum, tantalum, brass,sapphire, or any of the other numerous ceramic, metal, or polymericmaterials available in the art.

Ceramic plates are presently preferred because their inside surfaces maybe conveniently machined to very high smoothness for high wearresistance, high chemical resistance, and good thermal contact to theflexible walls of the reaction vessel. Ceramic plates can also be madevery thin, preferably between about 0.6 and 1.3 mm, for low thermal massto provide for extremely rapid temperature changes. A plate made fromceramic is also both a good thermal conductor and an electricalinsulator, so that the temperature of the plate may be well controlledusing a resistive heating element coupled to the plate.

Various thermal elements may be employed to heat and/or cool the plates50A, 50B and thus control the temperature of the reaction mixture in thechamber 17. In general, suitable heating elements for heating the plateinclude conductive heaters, convection heaters, or radiation heaters.Examples of conductive heaters include resistive or inductive heatingelements coupled to the plates, e.g., resistors or thermoelectricdevices.

Suitable convection heaters include forced air heaters or fluidheat-exchangers for flowing fluids past the plates. Suitable radiationheaters include infrared or microwave heaters. Similarly, variouscooling elements may be used to cool the plates. For example, variousconvection cooling elements may be employed such as a fan, peltierdevice, refrigeration device, or jet nozzle for flowing cooling fluidspast the surfaces of the plates. Alternatively, various conductivecooling elements may be used, such as a heat sink, e.g. a cooled metalblock, in direct contact with the plates.

Referring to FIG. 6, in the preferred embodiment, each plate 50 has aresistive heating element 56 disposed on its outer surface. Theresistive heating element 56 is preferably a thick or thin film and maybe directly screen printed onto each plate 50, particularly platescomprising a ceramic material, such as aluminum nitride or aluminumoxide. Screen-printing provides high reliability and low cross-sectionfor efficient transfer of heat into the reaction chamber. Thick or thinfilm resistors of varying geometric patterns may be deposited on theouter surfaces of the plates to provide more uniform heating, forexample by having denser resistors at the extremities and thinnerresistors in the middle. Although it is presently preferred to deposit aheating element on the outer surface of each plate, a heating elementmay alternatively be baked inside of each plate, particularly if theplates are ceramic. The heating element 56 may comprise metals,tungsten, polysilicon, or other materials that heat when a voltagedifference is applied across the material.

The heating element 56 has two ends which are connected to respectivecontacts 54 which are in turn connected to a voltage source (not shownin FIG. 6) to cause a current to flow through the heating element. Eachplate 50 also preferably includes a temperature sensor 52, such as athermocouple, thermistor, or RTD, which is connected by two traces 53 torespective contacts 54. The temperature sensor 52 may be used to monitorthe temperature of the plate 50 in a controlled feedback loop.

It is important that the plates have a low thermal mass to enable rapidheating and cooling of the plates. In particular, it is presentlypreferred that each of the plates has a thermal mass less than about 5J/° C., more preferably less than 3 J/° C., and most preferably lessthan 1 J/° C. As used herein, the term thermal mass of a plate isdefined as the specific heat of the plate multiplied by the mass of theplate. In addition, each plate should be large enough to cover arespective major wall of the reaction chamber. In the presentlypreferred embodiment, for example, each of the plates has a width X inthe range of 2 to 22 mm, a length Y in the range of 2 to 22 mm, and athickness in the range of 0.5 to 5 mm. The width X and length Y of eachplate is selected to be slightly larger than the width and length of thereaction chamber. Moreover, each plate preferably has an angled bottomportion matching the geometry of the bottom portion of the reactionchamber, as is described below with reference to FIG. 12. Also in thepreferred embodiment, each of the plates is made of aluminum nitridehaving a specific heat of about 0.75 J/g° C. The mass of each plate ispreferably in the range of 0.005 to 5.0 g so that each plate has athermal mass in the range of 0.00375 to 3.75 J/° C.

FIG. 8 is a schematic side view of a heat-exchanging module 60 intowhich the reaction vessel 12 is inserted for thermal processing andoptical interrogation. The module 60 preferably includes a housing 62for holding the various components of the module. The module 60 alsoincludes the thermally conductive plates 50 described above. The housing62 includes a slot (not shown in FIG. 8) above the plates 50 so that thereaction chamber of the vessel 12 may be inserted through the slot andbetween the plates. The heat-exchanging module 60 also preferablyincludes a cooling system, such as a fan 66. The fan 66 is positioned toblow cooling air past the surfaces of the plates 50 to cool the platesand hence cool the reaction mixture in the vessel 12. The housing 62preferably defines channels for directing the cooling air past theplates 50 and out of the module 60.

The heat-exchanging module 60 further includes an optical excitationassembly 68 and an optical detection assembly 70 for opticallyinterrogating the reaction mixture contained in the vessel 12. Theexcitation assembly 68 includes a first circuit board 72 for holding itselectronic components, and the detection assembly 68 includes a secondcircuit board 74 for holding its electronic components. The excitationassembly 68 includes one or more light sources, such as LEDs, forexciting fluorescently-labeled analytes in the vessel 12. The excitationassembly 68 also includes one or more lenses for collimating the lightfrom the light sources, as well as filters for selecting the excitationwavelength ranges of interest. The detection assembly 70 includes one ormore detectors, such as photodiodes, for detecting the light emittedfrom the vessel 12. The detection assembly 70 also includes one or morelenses for focusing and collimating the emitted light, as well asfilters for selecting the emission wavelength ranges of interest. Thespecific components of the optics assemblies 68, 70 are described ingreater detail below with reference to FIGS. 16-19.

The optics assemblies 68, 70 are positioned in the housing 62 such thatwhen the chamber of the vessel 12 is inserted between the plates 50, thefirst optics assembly 68 is in optical communication with the chamber 17through the optically transmissive side wall 19A (see FIG. 2) and thesecond optics assembly 70 is in optical communication with the chamberthrough the optically transmissive side wall 19B (FIG. 2). In thepreferred embodiment, the optics assemblies 68, 70 are placed intooptical communication with the optically transmissive side walls bysimply locating the optics assemblies 68, 70 next to the bottom edges ofthe plates 50 so that when the chamber of the vessel is placed betweenthe plates, the optics assemblies 68, 70 directly contact, or are inclose proximity to, the side walls.

As shown in FIG. 12, the vessel 12 preferably has an angled bottomportion (e.g., triangular) formed by the optically transmissive sidewalls 19A, 19B. Each of the plates 50A, 50B has a correspondingly shapedbottom portion. The bottom portion of the first plate 50A has a firstbottom edge 98A and a second bottom edge 98B. Similarly, the bottomportion of the second plate 50B has a first bottom edge 99A and a secondbottom edge 99B. The first and second bottom edges of each plate arepreferably angularly offset from each other by the same angle that theside walls 19A, 19B are offset from each other (e.g., 90°).Additionally, the plates 50A, 50B are preferably positioned to receivethe chamber of the vessel 12 between them such that the first side wall19A is positioned substantially adjacent and parallel to each of thefirst bottom edges 98A, 99A and such that the second side wall 19B ispositioned substantially adjacent and parallel to each of the secondbottom edges 98B, 99B. This arrangement provides for easy optical accessto the optically transmissive side walls 19A, 19B and hence to thechamber of the vessel 12.

The side walls 19A, 19B may be positioned flush with the edges of theplates 50A, 50B, or more preferably, the side walls 19A, 19B may bepositioned such that they protrude slightly past the edges of theplates. As is explained below with reference to FIGS. 16-19, each opticsassembly preferably includes a lens that physically contacts arespective one of the side walls 19A, 19B. It is preferred that the sidewalls 19A, 19B protrude slightly (e.g., 0.02 to 0.3 mm) past the edgesof the plates 50A, 50B so that the plates do not physically contact anddamage the lenses. A gel or fluid may optionally be used to establish orimprove optical communication between each optics assembly and the sidewalls 19A, 19B. The gel or fluid should have a refractive index close tothe refractive indexes of the elements that it is coupling.

Referring again to FIG. 8, the optics assemblies 68, 70 are preferablyarranged to provide a 90° angle between excitation and detection paths.The 90° angle between excitation and detection paths assures that aminimum amount of excitation radiation entering through the first sidewall of the chamber exits through the second side wall. Also, the 90°angle permits a maximum amount of emitted radiation to be collectedthrough the second side wall. In the preferred embodiment, the vessel 12includes a locating tab 27 (see FIG. 2) that fits into a slot formedbetween the optics assemblies 68, 70 to ensure proper positioning of thevessel 12 for optical detection. For improved detection, the module 60also preferably includes a light-tight lid (not shown) that is placedover the top of the vessel 12 and made light-tight to the housing 62after the vessel is inserted between the plates 50.

Although it is presently preferred to locate the optics assemblies 68,70 next to the bottom edges of the plates 50, many other arrangementsare possible. For example, optical communication may be establishedbetween the optics assemblies 68, 70 and the walls of the vessel 12 viaoptical fibers, light pipes, wave guides, or similar devices. Oneadvantage of these devices is that they eliminate the need to locate theoptics assemblies 68, 70 physically adjacent to the plates 50. Thisleaves more room around the plates in which to circulate cooling air orrefrigerant, so that cooling may be improved.

The heat-exchanging module 60 also includes a PC board 76 for holdingthe electronic components of the module and an edge connector 80 forconnecting the module 60 to a base instrument, as will be describedbelow with reference to FIG. 22. The heating elements and temperaturesensors on the plates 50, as well as the optical boards 72, 74, areconnected to the PC board 76 by flex cables (not shown in FIG. 8 forclarity of illustration). The module 60 may also include a groundingtrace 78 for shielding the optical detection circuit. The module 60 alsopreferably includes an indicator, such as an LED 64, for indicating to auser the current status of the module such as “ready to load sample”,“ready to load reagent,” “heating,” “cooling,” “finished,” or “fault”.

The housing 62 may be molded from a rigid, high-performance plastic, orother conventional material. The primary functions of the housing 62 areto provide a frame for holding the plates 50, optics assemblies 68, 70,fan 66, and PC board 76. The housing 62 also preferably provides flowchannels and ports for directing cooling air from the fan 66 across thesurfaces of the plates 50 and out of the housing. In the preferredembodiment, the housing 62 comprises complementary pieces (only onepiece shown in the schematic side view of FIG. 8) that fit together toenclose the components of the module 60 between them.

The opposing plates 50 are positioned to receive the chamber of thevessel 12 between them such that the flexible major walls of the chambercontact and conform to the inner surfaces of the plates. It is presentlypreferred that the plates 50 be held in an opposing relationship to eachother using, e.g., brackets, supports, or retainers. Alternatively, theplates 50 may be spring-biased towards each other as described inInternational Publication Number WO 98/38487, the disclosure of which isincorporated by reference herein. In another embodiment of theinvention, one of the plates is held in a fixed position, and the secondplate is spring-biased towards the first plate. If one or more springsare used to bias the plates towards each other, the springs should besufficiently stiff to ensure that the plates are pressed against theflexible walls of the vessel with sufficient force to cause the walls toconform to the inner surfaces of the plates.

FIGS. 9-10 illustrate a preferred support structure 81 for holding theplates 50A, 50B in an opposing relationship to each other. FIG. 9 showsan exploded view of the structure, and FIG. 10 shows an assembled viewof the structure. For clarity of illustration, the support structure 81and plates 50A, 50B are shown upside down relative to their normalorientation in the heat-exchanging module of FIG. 8. Referring to FIG.9, the support structure 81 includes a mounting plate 82 having a slot83 formed therein. The slot 83 is sufficiently large to enable thechamber of the vessel to be inserted through it. Spacing posts 84A, 84Bextend from the mounting plate 82 on opposite sides of the slot 83.Spacing post 84A has indentations 86 formed on opposite sides thereof(only one side visible in the isometric view of FIG. 9), and spacingpost 84B has indentations 87 formed on opposite sides thereof (only oneside visible in the isometric view of FIG. 9). The indentations 86, 87in the spacing posts are for receiving the edges of the plates 50A, 50B.To assemble the structure, the plates 50A, 50B are placed againstopposite sides of the spacing posts 84A, 84B such that the edges of theplates are positioned in the indentations 86, 87. The edges of theplates are then held in the indentations using a suitable retentionmeans. In the preferred embodiment, the plates are retained by retentionclips 88A, 88B. Alternatively, the plates 50A, 50B may be retained byadhesive bonds, screws, bolts, clamps, or any other suitable means.

The mounting plate 82 and spacing posts 84A, 84B are preferablyintegrally formed as a single molded piece of plastic. The plasticshould be a high temperature plastic, such as polyetherimide, which willnot deform of melt when the plates 50A, 50B are heated. The retentionclips 84A, 84B are preferably stainless steel. The mounting plate 82 mayoptionally include indentations 92A, 92B for receiving flex cables 90A,90B, respectively, that connect the heating elements and temperaturesensors disposed on the plates 50A, 50B to the PC board 76 of theheat-exchanging module 60 (FIG. 8). The portion of the flex cables 90Aadjacent the plate 50A is held in the indentation 92A by a piece of tape94A, and the portion of the flex cables 90B adjacent the plate 50B isheld in the indentation 92B by a piece of tape 94B.

FIG. 11 is an isometric view of the assembled support structure 81. Themounting plate 82 preferably includes tabs 96 extending from oppositesides thereof for securing the structure 81 to the housing of theheat-exchanging module. Referring again to FIG. 8, the housing 62preferably includes slots for receiving the tabs to hold the mountingplate 82 securely in place. Alternatively, the mounting plate 82 may beattached to the housing 62 using, e.g., adhesive bonding, screws, bolts,clamps, or any other conventional means of attachment.

Referring again to FIG. 9, the support structure 81 preferably holds theplates 50A, 50B so that their inner surfaces are angled very slightlytowards each other. In the preferred embodiment, each of the spacingposts 84A, 84B has a wall 89 that is slightly tapered so that when theplates 50A, 50B are pressed against opposite sides of the wall, theinner surfaces of the plates are angled slightly towards each other. Asbest shown in FIG. 5, the inner surfaces of the plates 50A, 50B angletowards each other to form a slightly V-shaped slot into which thechamber 17 is inserted. The amount by which the inner surfaces areangled towards each other is very slight, preferably about 1° fromparallel. The surfaces are angled towards each other so that, prior tothe insertion of the chamber 17 between the plates 50A, 50B, the bottomsof the plates are slightly closer to each other than the tops. Thisslight angling of the inner surfaces enables the chamber 17 of thevessel to be inserted between the plates and withdrawn from the platesmore easily. Alternatively, the inner surfaces of the plates 50A, 50Bcould be held parallel to each other, but insertion and removal of thevessel 12 would be more difficult.

In addition, the inner surfaces of the plates 50A, 50B are preferablyspaced from each other a distance equal to the thickness of the frame16. In embodiments in which the inner surfaces are angled towards eachother, the centers of the inner surfaces are preferably spaced adistance equal to the thickness of the frame 16 and the bottoms of theplates are initially spaced a distance that is slightly less than thethickness of the frame 16. When the chamber 17 is inserted between theplates 50A, 50B, the rigid frame 16 forces the bottom portions of theplates apart so that the chamber 17 is firmly sandwiched between theplates. The distance that the plates 50A, 50B are wedged apart by theframe 16 is usually very small, e.g., about 0.035 mm if the thickness ofthe frame is 1 mm and the inner surfaces are angled towards each otherby 1°.

Referring again to FIG. 10, the retention clips 88A, 88B should besufficiently flexible to accommodate this slight outward movement of theplates 50A, 50B, yet sufficiently stiff to hold the plates within therecesses in the spacing posts 84A, 84B during insertion and removal ofthe vessel. The wedging of the vessel between the plates 50A, 50Bprovides an initial preload against the chamber and ensures that theflexible major walls of the chamber, when pressurized, establish goodthermal contact with the inner surfaces of the plates.

Referring again to FIG. 8, to limit the amount that the plates 50 canspread apart due to the pressurization of the vessel 12, stops may bemolded into the housings of optics assemblies 68, 70. As shown in FIG.13, the housing 221 of the optics assembly 70 includes claw-like stops247A, 247B that extend outwardly from the housing. As shown in FIG. 14,the housing 221 is positioned such that the bottom edges of the plates50A, 50B are inserted between the stops 247A, 247B. The stops 247A, 247Bthus prevent the plates 50A, 50B from spreading farther than apredetermined maximum distance from each other. Although not shown inFIG. 14 for illustrative clarity, the optics assembly 68 (see FIG. 8)has a housing with corresponding stops for preventing the other halvesof the plates from spreading farther than the predetermined maximumdistance from each other. Referring again to FIG. 14, the maximumdistance that stops 247A, 247B permit the inner surfaces of the plates50A, 50B to be spaced from each other should closely match the thicknessof the frame 16. Preferably, the maximum spacing of the inner surfacesof the plates 50A, 50B is slightly larger than the thickness of theframe 16 to accommodate tolerance variations in the vessel 12 and plates50A, 50B. For example, the maximum spacing is preferably about 0.1 to0.3 mm greater than the thickness of the frame 16.

FIGS. 15A and 15B show the fluorescent excitation and emission spectra,respectively, of four fluorescent dyes of interest. These dyes arestandard fluorescent dyes used with the TaqMan® chemistry (availablefrom the Perkin-Elmer Corporation, Foster City, Calif.) and are wellknown by their acronyms FAM, TET, TAMRA, and ROX. Although the preferredembodiment is described with reference to these four dyes, it is to beunderstood that the apparatus of the present invention is not limited tothese particular dyes or to the TaqMang® chemistry. The apparatus may beused with any fluorescent dyes or with interculating dyes such asSYBRGreen™ or ethidium bromide. Such dyes are commercially availablefrom various well known suppliers. Fluorescent dyes and labelingchemistries for labeling analytes in a reaction mixture are well knownin the art and need not be discussed further herein. Further, althoughfluorescence detection is presently preferred, the apparatus of thepresent invention is not limited to detection based upon fluorescentlabels. The apparatus may be applicable to detection based uponphosphorescent labels, chemiluminescent labels, orelectrochemiluminescent labels.

As shown in FIG. 15A, the excitation spectra curves for FAM, TET, TAMRA,and ROX are typically very broad at the base, but sharper at the peaks.As shown in FIG. 15B, the relative emission spectra curves for the samedyes are also very broad at the base and sharper at the peaks. Thus,these dyes have strongly overlapping characteristics in both theirexcitation and emission spectra. The overlapping characteristics havetraditionally made it difficult to distinguish the fluorescent signal ofone dye from another when multiple dyes are used to label differentanalytes in a reaction mixture.

According to the present invention, multiple light sources are used toprovide excitation beams to the dyes in multiple excitation wavelengthranges. Each light source provides excitation light in a wavelengthrange matched to the peak excitation range of a respective one of thedyes. In the preferred embodiment, the light sources are blue and greenLEDs. FIG. 15C shows the effects of filtering the outputs of blue andgreen LEDs to provide substantially distinct excitation wavelengthranges. Typical blue and green LEDs have substantial overlap in therange of around 480 nm through 530 nm. By the filtering regime of thepresent invention, the blue LED light is filtered to a range of about450 to 495 nm to match the relative excitation peak for FAM. The greenLED light is filtered to a first range of 495 to 527 nm corresponding tothe excitation peak for TET, a second range of 527 to 555 nmcorresponding to the excitation peak for TAMRA, and a third range of 555to 593 nm corresponding to the excitation peak for ROX.

FIG. 15D shows the effects of filtering light emitted (fluorescentemission) from each of the four dyes to form distinct emissionwavelength ranges. As shown previously in FIG. 15B, the fluorescentemissions of the dyes before filtering are spherically diffuse withoverlapping spectral bandwidths, making it difficult to distinguish thefluorescent output of one dye from another. As shown in FIG. 15D, byfiltering the fluorescent emissions of the dyes into substantiallydistinct wavelength ranges, a series of relatively narrow peaks(detection windows) are obtained, making it possible to distinguish thefluorescent outputs of different dyes, thus enabling the detection of anumber of different fluorescently-labeled analytes in a reactionmixture.

FIG. 16 is a schematic, plan view of the optical excitation assembly 68.The assembly 68 is positioned adjacent the reaction vessel 12 totransmit excitation beams to the reaction mixture contained in thechamber 17. FIG. 17 is an exploded view of the excitation assembly. Asshown in FIGS. 16-17, the excitation assembly 68 includes a housing 219for holding various components of the assembly. The housing 219 includesstops 245A, 245B for limiting the maximum spacing of the thermal plates,as previously discussed with reference to FIGS. 8 and 14. The housing219 preferably comprises one or more molded pieces of plastic. In thepreferred embodiment, the housing 219 is a multi-part housing comprisedof three housing elements 220A, 220B, and 220C. The upper and lowerhousing elements 220A and 220C are preferably complementary pieces thatcouple together and snap-fit into housing element 220B. In thisembodiment, the housing elements 220A and 220C are held together byscrews 214. In alternative embodiments, the entire housing 219 may be aone-piece housing that holds a slide-in optics package.

The lower housing element 220C includes an optical window 235 into whichis placed a cylindrical rod lens 215 for focusing excitation beams intothe chamber 17. In general, the optical window 235 may simply comprisean opening in the housing through which excitation beams may betransmitted to the chamber 17. The optical window may optionally includean optically transmissive or transparent piece of glass or plasticserving as a window pane, or as in the preferred embodiment, a lens forfocusing excitation beams. The lens 215 preferably directly contacts oneof the optically transmissive side walls of the chamber 17.

The optics assembly 68 also includes four light sources, preferably LEDs100A, 100B, 100C, and 100D, for transmitting excitation beams throughthe lens 215 to the reaction mixture contained in the chamber 17. Ingeneral, each light source may comprise a laser, a light bulb, or anLED. In the preferred embodiment, each light source comprises a pair ofdirectional LEDs. In particular, the four light sources shown in FIGS.16-17 are preferably a first pair of green LEDs 100A, a second pair ofgreen LEDs 100B, a pair of blue LEDs 100C, and a third pair of greenLEDs 100D. The LEDs receive power through leads 201 which are connectedto a power source (not shown in FIGS. 16-17). The LEDs are mounted tothe optical circuit board 72 which is attached to the back of thehousing element 220B so that the LEDs are rigidly fixed in the housing.The optical circuit board 72 is connected to the main PC board of theheat-exchanging module (shown in FIG. 8) via the flex cable 103.

The optics assembly 68 further includes a set of filters and lensesarranged in the housing 219 for filtering the excitation beams generatedby the LEDs so that each of the beams transmitted to the chamber 17 hasa distinct excitation wavelength range. As shown in FIG. 17, the lowerhousing element 220C preferably includes walls 202 that create separateexcitation channels in the housing to reduce potential cross-talkbetween the different pairs of LEDs. The walls 202 preferably includeslots for receiving and rigidly holding the filters and lenses. Thefilters and lenses may also be fixed in the housing by means of anadhesive used alone, or more preferably, with an adhesive used incombination with slots in the housing.

Referring to FIG. 16, the filters in the optics assembly 68 may beselected to provide excitation beams to the reaction mixture in thechamber 17 in any desired excitation wavelength ranges. The opticsassembly 68 may therefore be used with any fluorescent, phosphorescent,chemiluminescent, or electrochemiluminescent labels of interest. Forpurposes of illustration, one specific embodiment of the assembly 68will now be described in which the assembly is designed to provideexcitation beams corresponding to the peak excitation wavelength rangesFAM, TAMRA, TET, and ROX.

In this embodiment, a pair of 593 nm low pass filters 203 are positionedin front of green LEDs 100A, a pair of 555 nm low pass filters 204 arepositioned in front of green LEDs 100B, a pair of 495 nm low passfilters 205 are positioned in front of blue LEDs 100C, and a pair of 527nm low pass filters 206 are positioned in front of green LEDs 100D.Although it is presently preferred to position a pair of low passfilters in front of each pair of LEDs for double filtering of excitationbeams, a single filter may be used in alternative embodiments. Inaddition, a lens 207 is preferably positioned in front of each pair offilters for collimating the filtered excitation beams. The opticsassembly 68 also includes a 495 nm high pass reflector 208, a 527 nmhigh pass reflector 209, a mirror 210, a 555 nm low pass reflector 211,and a 593 nm low pass reflector 212. The reflecting filters and mirrors208-212 are angularly offset by 30° from the low pass filters 203-206.

The excitation assembly 68 transmits excitation beams to the chamber 17in four distinct excitation wavelength ranges as follows. When the greenLEDs 100A are activated, they generate an excitation beam that passesthrough the pair of 593 nm low pass filters 203 and through the lens207. The excitation beam then reflects off of the 593 nm low passreflector 212, passes through the 555 nm low pass reflector 211,reflects off of the 527 nm high pass reflector 209, and passes throughthe lens 215 into the reaction chamber 17.

The excitation beam from the LEDs 100A is thus filtered to a wavelengthrange of 555 to 593 nm corresponding to the peak excitation range forROX. When the green LEDs 100B are activated, they generate an excitationbeam that passes through the pair of 555 nm low pass filters 204,reflects off of the 555 nm low pass reflector 211, reflects off of the527 nm high pass reflector 209, and passes through the lens 215 into thereaction chamber 17. The excitation beam from LEDs 100B is thus filteredto a wavelength range of 527 to 555 nm corresponding to the peakexcitation range for TAMRA.

When the blue LEDs 100C are activated, they generate an excitation beamthat passes through the pair of 495 nm low pass filters 205, through the495 μm high pass reflector 208, through the 527 nm high pass reflector209, and through the lens 215 into the reaction chamber 17. Theexcitation beam from LEDs 100C is thus filtered to a wavelength below495 nm corresponding to the peak excitation range for FAM. When thegreen LEDs 100D are activated, they generate an excitation beam thatpasses through the pair of 527 nm low pass filters 206, reflects off ofthe mirror 210, reflects off of the 495 nm high pass reflector 208,passes through the 527 nm high pass reflector 209, and passes throughthe lens 215 into the reaction chamber 17. The excitation beam from LEDs100D is thus filtered to a wavelength range of 495 to 527 nmcorresponding to the peak excitation range for TET. In operation, theLEDs 100A, 100B, 100C, 100D are sequentially activated to excite thedifferent fluorescent labels contained in the chamber 17 with excitationbeams in substantially distinct wavelength ranges.

FIG. 18 is a schematic, plan view of the optical detection assembly 70.The assembly 70 is positioned adjacent the reaction vessel 12 to receivelight emitted from the chamber 17. FIG. 19 is an exploded view of thedetection assembly 70. As shown in FIGS. 18-19, the assembly 70 includesa housing 221 for holding various components of the assembly.

The housing 221 includes the stops 247A, 247B previously described withreference to FIGS. 13-14. The housing 221 preferably comprises one ormore molded plastic pieces. In the preferred embodiment, the housing 221is a multi-part housing comprised of upper and lower housing elements234A and 234B. The housing elements 234A, 234B are complementary, matingpieces that are held together by screws 214. In alternative embodiments,the entire housing 221 may be a one-piece housing that holds a slide-inoptics package.

The lower housing element 234B includes an optical window 237 into whichis placed a cylindrical rod lens 232 for collimating light emitted fromthe chamber 17. In general, the optical window may simply comprise anopening in the housing through which the emitted light may be received.The optical window may optionally include an optically transmissive ortransparent piece of glass or plastic serving as a window pane, or as inthe preferred embodiment, the lens 232 for collimating light emittedfrom the chamber 17. The lens 232 preferably directly contacts one ofthe optically transmissive side walls of the chamber 17.

The optics assembly 70 also includes four detectors 102A, 102B. 102C,and 102D for detecting light emitted from the chamber 17 that isreceived through the lens 232. In general, each detector may be aphotomultiplier tube, CCD, photodiode, or other known detector. In thepreferred embodiment, each detector is a PIN photodiode. The detectors102A, 102B. 102C, and 102D are preferably rigidly fixed in recessesformed in the lower housing element 234B. The detectors are electricallyconnected by leads 245 to the optical circuit board 74 (see FIG. 8)which is preferably mounted to the underside of the lower housingelement 234B.

The optics assembly 70 further includes a set of filters and lensesarranged in the housing 221 for separating light emitted from thechamber 17 into different emission wavelength ranges and for directingthe light in each of the emission wavelength ranges to a respective oneof the detectors. As shown in FIG. 19, the lower housing element 234Bpreferably includes walls 247 that create separate detection channels inthe housing, with one of the detectors positioned at the end of eachchannel. The walls 247 preferably include slots for receiving andrigidly holding the filters and lenses. The filters and lenses may alsobe rigidly fixed in the housing 221 by an adhesive used alone, or morepreferably, with an adhesive used in combination with slots in thehousing.

Referring to FIG. 18, the filters in the optics assembly 70 may beselected to block light emitted from the chamber 17 outside of anydesired emission wavelength ranges. The optics assembly 70 may thereforebe used with any fluorescent, phosphorescent, chemiluminescent, orelectrochemiluminescent labels of interest. For purposes ofillustration, one specific embodiment of the assembly 70 will now bedescribed in which the assembly is designed to detect light emitted fromthe chamber 17 in the peak emission wavelength ranges of FAM, TAMRA,TET, and ROX.

In this embodiment, the set of filters preferably includes a 515 nmSchott Glass® filter 222A positioned in front of the first detector102A, a 550 nm Schott Glass® filter 222B positioned in front of thesecond detector 102B, a 570 nm Schott Glass® filter 222C positioned infront of the third detector 102C, and a 620 nm Schott Glass® filter 222Dpositioned in front of the fourth detector 102D. These Schott Glass®filters are commercially available from Schott Glass Technologies, Inc.of Duryea, Pa. The optics assembly 70 also includes a pair of 505 nmhigh pass filters 223 positioned in front of the first detector 102A, apair of 537 nm high pass filters 224 positioned in front of the seconddetector 102B, a pair of 565 nm high pass filters 225 positioned infront of the third detector 102C, and a pair of 605 nm high pass filters226 positioned in front of the fourth detector 102D.

Although it is presently preferred to position a pair of high passfilters in front of each detector for double filtering of light, asingle filter may be used in alternative embodiments. In addition, alens 242 is preferably positioned in each detection channel between thepair of high pass filters and the Schott Glass® filter for collimatingthe filtered light. The optics assembly 70 further includes a 605 nmhigh pass reflector 227, a mirror 228, a 565 nm low pass reflector 229,a 537 nm high pass reflector 230, and a 505 nm high pass reflector 231.The reflecting filters and mirrors 227-231 are preferably angularlyoffset by 30° from the high pass filters 223-226. As shown in FIG. 19,the detection assembly 70 also preferably includes a first aperture 238positioned between each detector and Schott Glass® filter 222 and anaperture 240 positioned between each lens 242 and Schott Glass® filter222. The apertures 238, 240 reduce the amount of stray or off-axis lightthat reaches the detectors 102A, 102B, 102C, and 102D.

Referring again to FIG. 18, the detection assembly 70 detects lightemitted from the chamber 17 in four emission wavelength ranges asfollows. The emitted light passes through the lens 232 and strikes the565 nm low pass reflector 229. The portion of the light having awavelength in the range of about 505 to 537 nm (corresponding to thepeak emission wavelength range of FAM) reflects from the 565 nm low passreflector 229, passes through the 537 nm high pass reflector 230,reflects from the 505 nm high pass reflector 231, passes through thepair of 505 nm high pass filters 223, through the lens 242, through the515 nm Schott Glass® filter 222A, and is detected by the first detector102A. Meanwhile, the portion of the light having a wavelength in therange of about 537 to 565 nm (corresponding to the peak emissionwavelength range of TET) reflects from the 565 nm low pass reflector229, reflects from the 537 nm high pass reflector 230, passes throughthe pair of 537 nm high pass filters 224, through the lens 242, throughthe 550 nm Schott Glass® filter 222B, and is detected by the seconddetector 102B.

Further, the portion of the light having a wavelength in the range ofabout 565 to 605 nm (corresponding to the peak emission wavelength rangeof TAMRA) passes through the 565 nm low pass reflector 229, through the605 nm high pass reflector 227, through the pair of 565 nm high passfilters 225, through the lens 242, through the 570 nm Schott Glass®filter 222C, and is detected by the third detector 102C. The portion ofthe light having a wavelength over 605 nm (corresponding to the peakemission wavelength range of ROX) passes through the 565 nm low passreflector 229, reflects from the 605 nm high pass reflector 227,reflects from the mirror 228, passes through the pair of 605 nm highpass filters 226, through the lens 242, through the 620 nm Schott Glass®filter 222D, and is detected by the fourth detector 102D. In operation,the outputs of detectors 102A, 102B, 102C, and 102D are analyzed todetermine the concentrations of each of the differentfluorescently-labeled analytes contained in the chamber 17, as will bedescribed in greater detail below.

FIG. 20 shows a multi-site reactor system 106 according to the presentinvention. The reactor system 106 comprises a thermal cycler 108 and acontroller 112, such as a personal or network computer. The thermalcycler 108 includes a base instrument 110 for receiving multipleheat-exchanging modules 60 (previously described with reference to FIG.8). The base instrument 110 has a main logic board with edge connectors114 for establishing electrical connections to the modules 60. The baseinstrument 110 also preferably includes a fan 116 for cooling itselectronic components. The base instrument 110 may be connected to thecontroller 112 using any suitable data connection, such as a universalserial bus (USB), ethernet connection, or serial line. It is presentlypreferred to use a USB that connects to the serial port of controller112. Alternatively, the controller may be built into the base instrument110.

The term “thermal cycling” is herein intended to mean at least onechange of temperature, i.e. increase or decrease of temperature, in areaction mixture. Therefore, samples undergoing thermal cycling mayshift from one temperature to another and then stabilize at thattemperature, transition to a second temperature or return to thestarting temperature. The temperature cycle may be performed only onceor may be repeated as many times as required to study or complete theparticular chemical reaction of interest. Due to space limitations inpatent drawings, the thermal cycler 108 shown in FIG. 20 includes onlysixteen reaction sites provided by the sixteen heat-exchanging modules60 arranged in two rows of eight modules each. It is to be understood,however, that the thermal cycler can include any number of desiredreaction sites, i.e., it can be configured as a multi-hundred siteinstrument for simultaneously processing hundreds of samples.Alternatively, it may be configured as a small, hand held,battery-operated instrument having, e.g., 1 to 4 reaction sites.

Each of the reaction sites in the thermal cycler 108 is provided by arespective one of the heat-exchanging modules 60. The modules 60 arepreferably independently controllable so that different chemicalreactions can be run simultaneously in the thermal cycler 108. Thethermal cycler 108 is preferably modular so that each heat-exchangingmodule 60 can be individually removed from the base instrument 110 forservicing, repair, or replacement. This modularity reduces downtimesince all the modules 60 are not off line to repair one, and theinstrument 110 can be upgraded and enlarged to add more modules asneeded. The modularity of the thermal cycler 108 also means thatindividual modules 60 can be precisely calibrated, and module-specificschedules or corrections can be included in the control programs, e.g.,as a series of module-specific calibration or adjustment charts.

In embodiments in which the base instrument 110 operates on externalpower, e.g. 110 V AC, the instrument preferably includes two powerconnections 122, 124. Power is received though the first connection 122and output through the second connection 124. Similarly, the instrument110 preferably includes network interface inlet and outlet ports 118,120 for receiving a data connection through inlet port 118 andoutputting data to another base instrument through outlet port 120. Asshown in the block diagram of FIG. 21, this arrangement permits multiplethermal cyclers 108A, 108B, 108C, 108D to be daisy-chained from onecontroller 112 and one external power source 128.

FIG. 22 is a schematic, block diagram of the base instrument 110. Thebase instrument includes a power supply 134 for supplying power to theinstrument and to each module 60. The power supply 134 may comprise anAC/DC converter for receiving power from an external source andconverting it to direct current, e.g., for receiving 110V AC andconverting it to 12V DC. Alternatively, the power supply 134 maycomprise a battery, e.g., a 12V battery. The base instrument 110 alsoincludes a microprocessor or microcontroller 130 containing firmware forcontrolling the operation of the base instrument 110 and modules 60. Themicrocontroller 130 communicates through a network interface 132 to thecontroller computer via a USB. Due to current limitations of processingpower, it is currently preferred to include at least one microcontrollerin the base instrument per sixteen modules 60. Thus if the baseinstrument has a thirty-two module capacity, at least twomicrocontrollers should be installed in the instrument 110 to controlthe modules.

The base instrument 110 further includes a heater power source andcontrol circuit 136, a power distributor 138, a data bus 140, and amodule selection control circuit 142. Due to space limitations in patentdrawings, control circuit 136, power distributor 138, data bus 140, andcontrol circuit 142 are shown only once in the block diagram of FIG. 22.However, the base instrument 110 actually contains one set of these fourfunctional components 136, 138, 140, 142 for each heat-exchanging module60. Thus, in the embodiment of FIG. 22, the base instrument 110 includessixteen control circuits 136, power distributors 138, data buses 140,and control circuits 142. Similarly, the base instrument 110 alsoincludes a different edge connector 131 for connecting to each of themodules 60, so that the instrument includes sixteen edge connectors forthe embodiment shown in FIG. 22. The edge connectors are preferably 120pin card edge connectors that provide cableless connection from the baseinstrument 110 to each of the modules 60. Each control circuit 136,power distributor 138, data bus 140, and control circuit 142 isconnected to a respective one of the edge connectors and to themicrocontroller 130.

Each heater power and source control circuit 136 is a power regulatorfor regulating the amount of power supplied to the heating element(s) ofa respective one of the modules 60. The source control circuit 136 ispreferably a DC/DC converter that receives a +12V input from the powersupply 134 and outputs a variable voltage between 0 and −24V. Thevoltage is varied in accordance with signals received from themicrocontroller 130. Each power distributor 138 provides −5 v, +5V,+12V, and GND to a respective module 60. The power distributor thussupplies power for the electronic components of the module. Each databus 140 provides parallel and serial connections between themicrocontroller 130 and the digital devices of a respective one of themodules 60. Each module selection controller 94 allows themicrocontroller 130 to address an individual module 60 in order to reador write control or status information.

FIG. 23 is a schematic, block diagram of the electronic components of aheat-exchanging module 60. Each module includes an edge connector 80 forcableless connection to a corresponding edge connector of the baseinstrument. The module also includes heater plates 50A, 50B each havinga resistive heating element as described above. The plates 50A, SOB arewired in parallel to receive power input 146 from the base instrument.The plates 50A, SOB also include temperature sensors 52, e.g.thermistors, that output analog temperature signals to ananalog-to-digital converter 154. The converter 154 converts the analogsignals to digital signals and routes them to the microcontroller in thebase instrument through the edge connector 80. The heat-exchangingmodule also includes a cooling system, such as a fan 66, for cooling theplates 50A, SOB. The fan 66 receives power from the base instrument andis activated by switching a power switch 164. The power switch 164 is inturn controlled by a control logic block 162 that receives controlsignals from the microcontroller in the base instrument.

The module further includes four light sources, such as LEDs 100, forexcitation of labeled analytes in the reaction mixture and fourdetectors 102, preferably photodiodes, for detecting fluorescentemissions from the reaction mixture. The module also includes anadjustable current source 150 for supplying a variable amount of current(e.g., in the range of 0 to 30 mA) to each LED to vary the brightness ofthe LED. A digital-to-analog converter 152 is connected between theadjustable current source 150 and the microcontroller of the baseinstrument to permit the microcontroller to adjust the current sourcedigitally. The adjustable current source 150 may be used to ensure thateach LED has about the same brightness when activated. Due tomanufacturing variances, many LEDs have different brightnesses whenprovided with the same amount of current. The brightness of each LED maybe tested during manufacture of the heat-exchanging module andcalibration data stored in a memory 160 of the module. The calibrationdata indicates the correct amount of current to provide to each LED. Themicrocontroller reads the calibration data from the memory 160 andcontrols the current source 150 accordingly. The microcontroller mayalso control the current source 150 to adjust the brightness of the LEDs100 in response to optical feedback received from the detectors 102.

The module additionally includes a signal conditioning/gainselect/offset adjust block 156 comprised of amplifiers, switches,electronic filters, and a digital-to-analog converter. The block 156adjusts the signals from the detectors 102 to increase gain, offset, andreduce noise. The microcontroller in the base instrument controls block156 through a digital output register 158. The output register 158receives data from the microcontroller and outputs control voltages tothe block 156. The block 156 outputs the adjusted detector signals tothe microcontroller through the analog-to-digital converter 154 and theedge connector 80. The module also includes the memory 160, preferably aserial EEPROM, for storing data specific to the module, such ascalibration data for the LEDs 100, thermal plates 50A, 50B, andtemperature sensors 52, as well as calibration data for a deconvolutionalgorithm described in detail below.

Referring again to FIG. 20, the reactor system 106 may be configured formanual filling and pressurization of each reaction vessel 12 by a humanoperator. Manual use of the system is suitable for lower throughputembodiments. For higher throughput embodiments, however, the system 106preferably includes automated machinery, e.g., a pick-and-place machine,for filling and pressurizing each of the vessels 12.

FIG. 24 shows a schematic diagram of a pick-and-place machine 166 forautomatically filling and pressurizing a reaction vessel 12. The machine166 has a pipette head 168 for engaging a disposable pipette tip 170.The machine 166 also has controllable vacuum and pressure sources incommunication with the pipette head 168 for aspirating and dispensingfluids using the pipette tip 170. The vacuum and pressure sources maycomprise, e.g., one or more syringe pumps, compressed air sources,pneumatic pumps, vacuum pumps, or connections to external sources ofpressure.

As shown in FIG. 25, the pick-and-place machine 166 also has a roboticarm or machine tip 172 for picking and placing the plunger 22 into thechannel 28 of the reaction vessel 12. The machine tip 172 may optionallyinclude an alignment pin 174 for aligning the cap 36 of the plunger in adesired angular orientation with respect to the body of the vessel 12.The alignment pin 174 provides a convenient mechanism for rotating thecap to the desired orientation before inserting the plunger 22 into thechannel 28. As previously shown in FIG. 3, the cap 36 includes a taperedengagement aperture 46 for receiving and establishing a friction fitwith the machine tip. The cap 36 also includes alignment apertures 48A,48B, either one of which may receive the alignment pin. Referring againto FIG. 25, the pick-and-place machine 166 also preferably includes anejector plate 176 that slides down the machine tip 172 to eject theplunger 22 from the machine tip after the plunger is inserted into thechannel 28. Although this embodiment of the pick-and-place machine ispresently preferred, many other embodiments are possible. For example,the machine tip 172 may be designed to establish a vacuum fit with thecap 36 of the plunger. Alternatively, the pick-and-place machine mayhave a robotic gripper arm for gripping the plunger 22 and inserting itinto the channel 28. Suitable pick-and-place machines for use in theapparatus of the present invention are commercially available asmachines built to specification from several suppliers, such as TecanU.S. Inc. located at 4022 Stirrup Creek Drive, Durham, N.C. 27703.

Referring again to FIG. 20, the controller 112 preferably includessoftware for controlling the thermal cycler 108 and the pick-and-placemachine (described above with reference to FIGS. 24-25) to perform thefunctions described in the operation section below. These functionsinclude providing a user interface to enable a user to select desiredthermal processing parameters (e.g., set point temperatures and holdtimes at each temperature) and optical detection parameters, automaticfilling and pressurization of the vessels 12, thermal processing of thevessels according to the selected parameters, optical interrogation ofthe reaction mixtures in the vessels, and recording of the optical datagenerated. The creation of software and/or firmware for performing thesefunctions can be performed by a computer programmer having ordinaryskill in the art. Moreover, the software and/or firmware may residesolely in the controller 112 or may be distributed between thecontroller and one or more microprocessors in the thermal cycler orpick-and-place machine. Alternatively, the controller 112 may simply bebuilt into the thermal cycler or pick-and-place machine.

In operation, the reactor system 106 is used to thermally process andoptically interrogate one or more samples. An exemplary use of thesystem 106 is for the amplification of nucleic acid in a sample (e.g.,using PCR) and for the optical detection of one or more target analytesin the sample. A user selects a desired thermal profile for the sampleusing, e.g., the keyboard or mouse of the controller 112. For example,for a PCR amplification, the user may select the thermal profile tobegin with a 30 second induction hold at 95° C., followed by 45 thermalcycles in which the reaction mixture is cycled between higher and lowertemperatures for denaturization, annealing, and polymerization. Forexample, each thermal cycle may include a first set point temperature of95° C. which is held for 1 second to denature double-stranded DNA,followed by a second set point temperature of 60° C. which is held for 6seconds for annealing of primers and polymerization.

Referring again to FIG. 24, the sample is preferably dispensed into thevessel 12 by aspirating the sample into the pipette tip 170, insertingthe pipette tip 170 through the channel 28 into the chamber 17, anddispensing the sample into the chamber. It is presently preferred thatthe chamber 17 be filled from the bottom up by initially inserting thepipette tip 170 close to the bottom of the chamber 17 and by slowlyretracting the pipette tip 170 as the chamber 17 is filled. Filling thechamber 17 in this manner reduces the likelihood that air bubbles willform in the chamber. Such air bubbles could have a negative effect onsubsequent optical detection.

The fluid sample may be mixed with chemicals necessary for the intendedreaction (e.g., PCR reagents and/or fluorescent probes) prior to beingadded to the chamber 17. Alternatively, the sample may be introduced tothe chemicals in the chamber 17, e.g., by adding the chemicals to thechamber before or after the sample to form the desired reaction mixturein the chamber. In a particularly advantageous embodiment, the necessaryreagents and/or fluorescent probes for the intended reaction are placedin the chamber 17 when the vessel is manufactured. The reagents arepreferably placed in the chamber 17 in dried or lyophilized form so thatthey are adequately preserved until the vessel is used.

Referring again to FIG. 25, the chamber 17 is then pressurized after itis filled with the reaction mixture. To increase pressure in the vessel,the machine tip 172 of the pick-and-place machine 166 engages the cap 36of the plunger 22 and inserts the plunger into the channel 28 until thesnap ring 38 snaps into the annular recess 23. As the plunger 22 isinserted, the piston 32 compresses gas in the channel 28 to increasepressure in the chamber 17, preferably to about 8 to 15 psi aboveambient pressure, as previously discussed with reference to FIGS. 7A-7D.After the plunger 22 is inserted, the ejector plate 176 ejects theplunger 22 from the machine tip 172.

Referring again to FIG. 20, each of the vessels 12 may be insertedbetween the thermal plates of a respective heat-exchanging module 60either prior to filling and pressurizing the vessel or after filling andpressurizing the vessel. In either case, as shown in FIG. 5, thepressure in the chamber 17 forces the flexible major walls 18 to contactand conform to the inner surfaces of the plates 50. In embodiments inwhich the vessels are inserted between the plates prior to filling andpressurization, the pick-and-place machine includes a robotic arm (notshown) for picking up the vessels and inserting them into the modules.Robotic arms for picking and placing reaction vessels are well known inthe art.

Referring again to FIG. 25, each vessel 12 may alternatively be insertedbetween the plates of a respective module after filling andpressurization using the machine tip 172. In this embodiment, the vessel12 is preferably held in a rack, tray, or similar support device duringfilling and pressurization. After the vessel 12 is filled andpressurized, the machine tip 172 picks up the vessel 12 by the cap 36 ofthe plunger 22 and inserts the chamber 17 of the vessel between theplates of a heat-exchanging module. The plunger 22 is held in thechannel 28 during this movement by the snap ring 38 that engages theannular recess 23. After the vessel 12 is inserted, the ejector plate176 ejects the cap 36 from the machine tip 172.

Although automated filling and pressurization of the vessel 12 has beendescribed, the vessel may also be manually filled and pressurized by ahuman operator. This is most easily accomplished by filling the chamber17 using a hand-held pipette or syringe and by manually inserting theplunger 22 into the channel 28. The operator then inserts the chamber 17of the vessel into one of the heat-exchanging modules.

Referring again to FIG. 20, once a filled and pressurized reactionvessel 12 is placed between the thermal plates of a heat-exchangingmodule 60, the reaction mixture contained in the vessel is subjected tothe thermal profile selected by the user. The controller 112 preferablyimplements standard proportional-integral-derivative (PID) control toexecute the selected thermal profile. Referring again to FIG. 23, thecontroller receives signals indicating the temperatures of the plates50A, 50B from the temperature sensors 52. Polling of the platetemperatures preferably occurs every 100 milliseconds throughout therunning of the temperature profile. After each polling, the controlleraverages the temperatures of the two plates 50A, 50B to determine anaverage plate temperature. The controller then determines the difference(delta) between the profile target temperature, i.e. the set pointtemperature defined by the user for the particular time in the profile,and the average plate temperature. Based on the relationship between theaverage plate temperature and the current target temperature, thecontroller controls the amount of power supplied to the heating elementson the plates 50A, 50B or to the fan 66 as appropriate to reach ormaintain the current set point temperature. Standard PID control is wellknown in the art and need not be described further herein.

The controller may optionally implement a modified version of PIDcontrol described in International Publication Number WO 99/48608published Sep. 30, 1999, the disclosure of which is incorporated byreference herein. In this modified version of PID control, thecontroller is programmed to compensate for thermal lag between theplates 50A, 50B and a reaction mixture contained in a reaction vesselinserted between the plates. The thermal lag is caused by the need forheat to transfer from the plates 50A, 50B through the flexible walls ofthe vessel and into the reaction mixture during heating, or by the needfor heat to transfer from the reaction mixture through the walls of thevessel to the plates 50A, 50B during cooling. In standard PID control,the power supplied to a heating or cooling element is dependent upon thedifference (error) between the actual measured temperature of the platesand the desired set point temperature. The average power being suppliedto either the heating or cooling element therefore decreases as theactual temperature of the plates approaches the set point temperature,so that the reaction mixture does not reach the set point temperature asrapidly as possible. The modified version of PID control overcomes thisdisadvantage of standard PID control during rapid heating or coolingsteps.

To compensate for the thermal lag during heating steps (i.e., to raisethe temperature of the reaction mixture to a desired set pointtemperature that is higher than the previous set point temperature), thecontroller sets a variable target temperature that initially exceeds thedesired set point temperature. For example, if the set point temperatureis 95° C., the initial value of the variable target temperature may beset 2 to 10° C. higher. The controller next determines a level of powerto be supplied to the heating elements to raise the temperature of theplates 50A, 50B to the variable target temperature by inputting thevariable target temperature and the current average plate temperature toa standard PID control algorithm. The level of power to be supplied tothe heaters is therefore determined in dependence upon the difference(error) between the average plate temperature and a target temperaturethat is higher than the desired set point temperature. The higher targettemperature ensures that a higher level of power is supplied to heat theplates 50A, 50B, and therefore the reaction mixture, to the set pointtemperature more rapidly. The controller then sends a control signal tothe power and source control circuit in the base instrument to providepower to the heating elements at the level determined.

When the temperature of the plates 50A, 50B is subsequently polled, thecontroller determines if the actual measured temperature of the platesis greater than or equal to a predetermined threshold value. Suitablethreshold values are: the desired set point temperature itself; or 1 to2° C. below the set point temperature, e.g., 93 to 94° C. for a setpoint temperature of 95° C. If the average plate temperature does notexceed the predetermined threshold value, then the controller againdetermines a level of power to be supplied to the heating elements independence upon the difference between the average plate temperature andthe target temperature and sends another control signal to provide powerto the heaters at the level determined. This process is repeated untilthe average plate temperature is greater than or equal to the thresholdvalue.

When the average plate temperature is greater than or equal to thethreshold value, the controller decreases the variable targettemperature, preferably by exponentially decaying the amount by whichthe variable target temperature exceeds the set point temperature. Forexample, the amount by which the variable target temperature exceeds thedesired set point temperature may be exponentially decayed as a functionof time according to the equation:Δ=(Δ_(max))*e ^((−t/tau))where Δ is equal to the amount by which the variable target temperatureexceeds the desired set point temperature, Δ_(max) is equal to thedifference between the initial value of the variable target temperatureand the desired set point temperature, t is equal to the elapsed time inseconds from the start of decay, and tau is equal to a decay timeconstant.

In the system of the present invention, tau preferably has a value inthe range of 1 to 4 seconds. It is presently preferred to determine tauempirically for the heat-exchanging module during testing andcalibration of the module and to store the value of tau in the memory160 of the module before shipping it to the end user. Although theexponential equation given above is presently preferred, it is to beunderstood that many other decay formulas may be employed and fallwithin the scope of the invention. Moreover, the variable targettemperature may be decreased by other techniques, e.g., it may bedecreased linearly.

After decreasing the variable target temperature, the controllerdetermines a new level of power to be supplied to the heating elementsto raise the temperature of the plates 50A, SOB to the decreased targettemperature. The controller determines the level of power by inputtingthe current plate temperature and decreased target temperature to thePID control algorithm. The controller then sends a control signal toprovide power to the heaters at the new level determined. As the time inthe thermal profile progresses, the controller continues to decrease thevariable target temperature until it is equal to the set pointtemperature. When the variable target temperature is equal to the setpoint temperature, standard PID control is resumed to maintain theplates 50A, SOB at the set point temperature.

To compensate for the thermal lag during cooling steps (i.e., to lowerthe temperature of the reaction mixture to a desired set pointtemperature that is lower than the previous set point temperature), thecontroller preferably activates the fan 66 just prior to the completionof the previous set point temperature to allow the fan to achievemaximum speed for cooling (i.e., to allow for spin-up time). Thecontroller then sets a variable target temperature that is initiallylower than the desired set point temperature. For example, if the setpoint temperature is 60° C., the initial value of the variable targettemperature may be set 2 to 10° C. lower, i.e., 50 to 58° C. Thecontroller continues cooling with the fan 66 until the actual measuredtemperature of the plates 50A, SOB is less than or equal to a thresholdvalue, preferably the variable target temperature. When the averageplate temperature is less than or equal to the variable targettemperature, the controller deactivates the fan 66 and increases thetarget temperature, preferably by exponentially decaying the amount bywhich the variable target temperature differs from the set pointtemperature using the exponential decay equation given above. Forcooling, tau is preferably in the range of 1 to 5 seconds with apreferred value of about 3 seconds. As in the heating example givenabove, tau may be determined empirically for the heat-exchanging moduleduring testing or calibration and stored in the memory 160.

The controller next determines a level of power to be supplied to theheating elements to raise the temperature of the plates 50A, SOB to theincreased target temperature by inputting the current average platetemperature and the increased target temperature to the PID controlalgorithm. The controller then sends a control signal to the power andsource control circuit in the base instrument to provide power to theheating elements at the level determined. As time in the thermal profilecontinues, the controller continues to increase the variable targettemperature and issue control signals in this manner until the variabletarget temperature is equal to the set point temperature. When thevariable target temperature is equal to the set point temperature, thecontroller resumes standard PID control to maintain the plates 50A, SOBat the set point temperature.

Referring again to FIGS. 16 and 18, the reaction mixture in the vessel12 is optically interrogated in real-time as the thermal profile isexecuted to determine if the mixture contains one or more targetanalytes. In the preferred embodiment, the mixture is opticallyinterrogated once per thermal cycle at the lowest temperature in thecycle. Optical interrogation is accomplished by sequentially activatingLEDs 100A, 100B, 100C, and 100D to excite differentfluorescently-labeled analytes in the mixture and by detecting lightemitted (fluorescent output) from the chamber 17 using detectors 102A,102B, 102C, and 102D. In the following example of operation, thefluorescent dyes FAM, TAMRA, TET, and ROX are used to label the targetanalytes, e.g., target nucleotide sequences, nucleic acids, proteins,pathogens, or organisms in the reaction mixture.

There are four pairs of LEDs 100A, 100B, 100C, and 100D and fourdetectors 102A, 102B, 102C, and 102D for a total of sixteen combinationsof LED/detector pairs. It is theoretically possible to collect outputsignals from the detectors for all sixteen combinations. Of thesesixteen combinations, however, there are only four primary detectionchannels. Each primary detection channel is formed by a pair of LEDs inthe optics assembly 68 whose excitation beams lie in the peak excitationwavelength range of a particular dye and by one corresponding detectionchannel in the optics assembly 70 designed to detect light emitted inthe peak emission wavelength range of the same dye. The first primarydetection channel is formed by the first pair of LEDs 100A and thefourth detector 102D (the ROX channel). The second primary detectionchannel is formed by the second pair of LEDs 100B and the third detector102C (the TAMRA channel). The third primary detection channel is formedby the third pair of LEDs 100C and the first detector 102A (the FAMchannel). The fourth primary detection channel is formed by the fourthpair of LEDs 100D and the second detector 102B (the TET channel).

Prior to activating any of the LEDs 100A, 100B, 100C, 100D, a “darkreading” is taken to determine the output signal of each of the fourdetectors 102A, 102B, 102C, 102D when none of the LEDs are lit. The“dark reading” signal output by each detector is subsequently subtractedfrom the corresponding “light reading” signal output by the detector tocorrect for any electronic offset in the optical detection circuit. Thisprocedure of obtaining “dark reading” signals and subtracting the darksignals from the corresponding “light reading” signals is preferablyperformed every time that a reaction vessel is optically interrogated,including those times the vessel is interrogated during the developmentof calibration data (described in detail below). For clarity and brevityof explanation, however, the steps of obtaining “dark reading” signalsand subtracting the dark signals from the corresponding “light reading”signals will not be further repeated in this description.

Following the dark reading, a “light reading” is taken in each of thefour primary optical detection channels as follows. The first pair ofLEDs 100A is activated and the LEDs generate an excitation beam thatpasses through the pair of 593 nm low pass filters 203, reflects off ofthe 593 nm low pass reflector 212, passes through the 555 nm low passreflector 211, reflects off of the 527 nm high pass reflector 209, andpasses through the lens 215 into the reaction chamber 17. The excitationbeam from the LEDs 100A is thus filtered to a wavelength range of 555 to593 nm corresponding to the peak excitation range for ROX. As shown inFIG. 18, emitted light (fluorescence emission radiation) from thechamber 17 passes through the lens 232 of the detection assembly 70 andstrikes the 565 nm low pass reflector 229. The portion of the lighthaving a wavelength over 605 nm (corresponding to the peak emissionwavelength range of ROX) passes through the 565 nm low pass reflector229, reflects from the 605 nm high pass reflector 227, reflects from themirror 228, passes through the pair of 605 nm high pass filters 226,through the lens 242, through the 620 nm Schott Glass® filter 222D, andis detected by the fourth detector 102D. The fourth detector 102Doutputs a corresponding signal that is converted to a digital value andrecorded.

Next, as shown in FIG. 16, the second pair of LEDs 100B is activated andthe LEDs generate an excitation beam that passes through the pair of 555nm low pass filters 204, reflects off of the 555 nm low pass reflector211, reflects off of the 527 nm high pass reflector 209, and passesthrough the lens 215 into the reaction chamber 17. The excitation beamfrom LEDs 100B is thus filtered to a wavelength range of 527 to 555 nmcorresponding to the peak excitation range for TAMRA. As shown in FIG.18, emitted light from the chamber 17 then passes through the lens 232of the detection assembly 70 and strikes the 565 nm low pass reflector229. The portion of the light having a wavelength in the range of about565 to 605 nm (corresponding to the peak emission wavelength range ofTAMRA) passes through the 565 nm low pass reflector 229, through the 605nm high pass reflector 227, through the pair of 565 nm high pass filters225, through the lens 242, through the 570 nm Schott Glass® filter 222C,and is detected by the third detector 102C. The third detector 102Coutputs a corresponding signal that is converted to a digital value andrecorded.

Next, as shown in FIG. 16, the pair of blue LEDs 100C is activated andthe LEDs generate an excitation beam that passes through the pair of 495nm low pass filters 205, through the 495 nm high pass reflector 208,through the 527 nm high pass reflector 209, and through the lens 215into the reaction chamber 17. The excitation beam from LEDs 100C is thusfiltered to a wavelength range of about 450 to 495 nm corresponding tothe peak excitation range for FAM. As shown in FIG. 18, emitted lightfrom the chamber 17 then passes through the lens 232 of the detectionassembly 70 and strikes the 565 nm low pass reflector 229. The portionof the light having a wavelength in the range of about 505 to 537 nm(corresponding to the peak emission wavelength range of FAM) reflectsfrom the 565 nm low pass reflector 229, passes through the 537 nm highpass reflector 230, reflects from the 505 nm high pass reflector 231,passes through the pair of 505 nm high pass filters 223, through thelens 242, through the 515 nm Schott Glass® filter 222A, and is detectedby the first detector 102A. The first detector 102A outputs acorresponding signal that is converted to a digital value and recorded.

Next, as shown in FIG. 16, the fourth pair of LEDs 100D is activated andthe LEDs generate an excitation beam that passes through the pair of 527nm low pass filters 206, reflects off of the mirror 210, reflects off ofthe 495 nm high pass reflector 208, passes through the 527 nm high passreflector 209, and passes through the lens 215 into the reaction chamber17. The excitation beam from LEDs 100D is thus filtered to a wavelengthrange of 495 to 527 nm corresponding to the peak excitation range forTET. As shown in FIG. 18, emitted light from the chamber 17 then passesthrough the lens 232 of the detection assembly 70 and strikes the 565 nmlow pass reflector 229. The portion of the light having a wavelength inthe range of about 537 to 565 nm (corresponding to the peak emissionwavelength range of TET) reflects from the 565 nm low pass reflector229, reflects from the 537 nm high pass reflector 230, passes throughthe pair of 537 nm high pass filters 224, through the lens 242, throughthe 550 nm Schott Glass® filter 222B, and is detected by the seconddetector 102B. The second detector 102B outputs a corresponding signalthat is converted to a digital value and recorded. The total timerequired to activate each of the four LEDs 100A, 100B, 100C, 100D insequence and to collect four corresponding measurements from thedetectors 102A, 102B, 102C, 102D is typically five seconds or less.

The spectrum of the fluorescence that is emitted by the dyes used fordetection is usually broad. As a result, when an individual dye (e.g.,FAM, TAMRA, TET, or ROX) emits fluorescence from the reaction vessel 12,the fluorescence can be detected in several of the primary detectionchannels, i.e. several of the detectors 102A, 102B, 102C, and 102Ddetect the fluorescence and generate an output signal. However, each dyehas its own ‘signature’, i.e., the ratios of the optical signals in eachdetection channel are unique to each dye. It is also a reasonableassumption that the fluorescent emission from a mixture of dyes aresimply additive in each of the detection channels, so that theindividual dye concentrations of a dye mixture can be extracted from themixed signals using linear algebra.

In the preferred embodiment, the controller is programmed to convert theoutput signals of the detectors to values indicating the trueconcentration of each dye labeling a respective analyte in the reactionmixture using linear algebra and a calibration matrix. A preferredmethod for developing the calibration matrix will now be described usingthe four-channel optical system of the preferred embodiment as anexample. First, a reaction vessel containing only reaction buffer isoptically read using optics assemblies 68, 70. The reaction buffershould be a fluid similar or nearly identical to the reaction mixturesthat will be optically read by the optics assemblies during actualproduction use of the system to test samples. The reaction buffer shouldcontain no dyes, so that the concentrations of all dyes are zero. Theoptical reading of the reaction buffer in the four primary detectionchannels produces four output signals that are converted tocorresponding digital values. These four numbers are called Buffer(I),where ‘I’ is 1, 2, 3 or 4 depending upon which detection channel isread. The buffer values are a measure of the background signal orscattered light detected in each primary detection channel without anyadded fluorescent signal from dyes.

Next, a reaction mixture containing a known concentration, e.g. 100 nM,of dye #1 is placed into the vessel and again the four channels areread. The four numbers produced are called Rawdye(I, 1). Similar sets offour numbers are obtained for the other three dyes to obtain Rawdye(I,2), Rawdye(I, 3), and Rawdye(I, 4). The buffer values are thensubtracted from the raw dye values to obtain net dye values as follows:Netdye(I,J)=Rawdye(I,J)−Buffer(I);where I indicates the detection channel, and J indicates the dye number.

The matrix Netdye(I, J) is then inverted using standard numericalmethods (such as Gaussian elimination) to obtain a new matrix called thecalibration matrix Cal(I,J). Note that the matrix product of Netdye(I,J)*Cal(I,J) is the unity matrix. Now, any reaction mixture can be readand the output signals of the detectors in the four detection channelsconverted to values representative of the true concentrations of dyeslabeling analytes in the mixture. The optical reading of the mixtureproduces four numbers called RawMix(I). The reaction buffer values arethen subtracted from the raw mix values to obtain four numbers calledMix(I) as follows:Mix(I)=RawMix(I)−Buffer(I)

Next, the true concentrations of the dyes labeling analytes are obtainedby matrix multiplication as follows:Truedye(I)=100 nM*Cal(I,J)*Mix(I)

In the above equation, the factor of 100 comes from the fact that aconcentration of 100 nM was used for the initial calibrationmeasurements. The concentration of 100 nM is used for purposes ofexample only and is not intended to limit the scope of the invention.

In general, the dye concentrations for calibration measurements shouldbe somewhere in the range of 25 to 1,000 mM depending upon thefluorescent efficiency (strength) of the dyes and their use in aparticular assay or application.

Referring again to FIGS. 22-23, the matrices Cal(I, J) and Buffer(I) arepreferably produced during the manufacture of each heat-exchangingmodule 60 and stored in the memory 160. When the module 60 is pluggedinto the base instrument 110, the control software application in thebase instrument or external computer reads the matrices into memory anduses the matrices to convert the output signals of the detectors 102 tovalues indicating the concentration of each dye in the reaction mixture.Because the calibration matrices Cal(I, J) and Buffer(I) are dependentupon the particular set of dyes calibrated and the volume of thereaction vessel, it is also preferred to produce and store multiple setsof the matrices for various combinations of dye sets and reaction vesselvolumes. This gives the end user greater flexibility in using thesystem.

As one example, calibration matrices could be stored for three differentdye sets to be used with three different sizes of reaction vessels(e.g., 25 μl, 50 μl, 100 μl) for a total of nine different sets ofcalibration matrices. Of course, this is just one example, and manyother combinations will be apparent to one skilled in the art uponreading this description. Further, in alternative embodiments, thecontrol software may include functionality to guide the end user throughthe calibration procedure to enable the user to store and usecalibration data for his or her own desired combination of dyes andreaction vessel size.

It is presently preferred to perform an optical reading of the reactionmixture once per thermal cycle at the lowest temperature in the cycle.Alternatively, the reaction mixture could be optically monitored morefrequently or less frequently as desired by the user. One advantage tofrequent optical monitoring is that real-time optical data may be usedto indicate the progress of the reaction. For example, when a particularpredetermined fluorescent threshold is detected in a reaction mixture ina heat-exchanging module, then the temperature cycling for that modulemay be stopped. Furthermore, optical detection of dye activation, e.g.,color change, is useful to control the cycle parameters, not onlythermal schedules, but also the state or condition of reactants andproducts, and quantitative production. Multiple emission wavelengths canbe sampled to determine, for example, progression of the reaction, endpoints, triggers for reagent addition, denaturization (melting),annealing and the like. The data obtained in the real-time monitoringmethod may be fed back to the controller to alter or adjust the optical“read” parameters. Examples of the optical read parameters include:length of read; power input or frequency to the LEDs; which wavelengthshould be monitored and when; and the like.

In a typical implementation of the four-channel system, three of theoptical channels are used to detect target analytes (e.g., amplifiednucleic acid sequences) while the fourth channel is used to monitor aninternal control to check the performance of the system. For example,beta actin is often used as an internal control in nucleic acidamplification reactions because it has a predictable amplificationresponse and can be easily labeled and monitored to verify that theamplification is occurring properly.

One advantage of the apparatus of the preferred embodiment is that itprovides extremely rapid heating and cooling of a reaction mixture. Thisrapid heating and cooling is particularly beneficial for nucleic acidamplification because of the increased speed with which theamplification may be accomplished and because it significantly reducesthe likelihood of creating unwanted and interfering side products, suchas PCR “primer-dimers” or anomalous amplicons. Another advantage of theapparatus is that it provides for sensitive, real-time detection of oneor more analytes in a reaction mixture as the reaction is performed. Inexperimental testing of the apparatus of the preferred embodiment,extraordinary results for nucleic acid amplification and detection wereachieved. For example, a 100 μl sample containing bacillus globigii in astarting concentration of 105 copies per ml has been amplified anddetected in about 8 minutes (24 thermal cycles having a duration of 21seconds per cycle).

FIG. 26 shows a reaction vessel 180 according to another embodiment ofthe invention. The vessel 180 is similar to the vessel of the preferredembodiment (described with reference to FIGS. 1-2), except that thevessel 180 has a smaller reaction chamber 184. The size of the chamber184 is defined by the side walls 186A, 186B, 188A, 188B and by thethickness of the rigid frame 182. In this embodiment, each of the sidewalls 186A, 186B, 188A, 188B has a length L of about 5 mm, the chamberhas a width W of about 7 mm, and the chamber has a thickness T of 1 mmso that the chamber has a volume capacity of 25 μL. The advantage to thevessel 180 is that it holds a smaller volume of reaction mixture so thatthe mixture requires less reagent. The disadvantage is that the smallervolume may cause decreased sensitivity in the detection of lowconcentration analytes, such as nucleic acids. The vessel 180demonstrates that the reaction vessels of the present invention may befabricated with chambers having various volume capacities, preferably inthe range of 5 to 200 μl. It is presently preferred to fabricate each ofthe vessels with substantially the same size frame, regardless of thevolume capacity of the chamber, so that each of the vessels may be usedwith the same size heat-exchanging module 60 (FIG. 8).

FIG. 27 shows a reaction vessel 190 according to another embodiment ofthe invention. The vessel 190 is similar to the vessel of the preferredembodiment (described with reference to FIGS. 1-2), except that thevessel 190 has an elastomeric plunger 192. The plunger 192 isconstructed of an elastomeric material, e.g., a thermal plasticelastomer (TPE) or silicone. The elastomeric plunger 192 preferablyincludes a sealing ring 194 that establishes a seal with the walls ofthe channel 193 when the plunger is inserted into the channel tocompress gas in the vessel and increase pressure in the chamber 191. Theplunger 192 may be manually inserted into the channel 193 by humanhands, or alternatively, the plunger 192 may include an engagementaperture to permit a pick-and-place machine to pick and place theplunger into the channel.

FIG. 28 shows a reaction vessel 250 according to another embodiment ofthe invention. The vessel 250 is similar to the vessel of the preferredembodiment (described with reference to FIGS. 1-2), except that thevessel 250 has several additional features. In particular, the plunger252 of the vessel has a plunger cap 259 on which are formed ramp-shapedprotrusions 260A, 260B. The vessel includes corresponding ramp-shapedprotrusions 262A, 262B which are preferably formed on the finger grips26 and positioned on opposite sides of the port 14. The correspondingsets of ramp-shaped protrusions engage each other provide for easytwist-off of the plunger 252, if desired, after the vessel 250 is used.

The plunger 252 also includes a stem 254 that terminates in a tongue258. As shown in FIG. 29, the stem 254 has a length substantially equalto the length of the channel 28 so that the tip of the tongue 258 ispositioned at the end of the channel 28 adjacent the entrance 266 to thechamber 17 when the plunger 252 is fully inserted in the channel. Theadvantage of the tongue 258 is that it provides a physical barrier forpreventing the reaction mixture in the chamber 17 from bubbling up(refluxing) or evaporating into the channel 28 as the mixture is heated.As shown in FIG. 30, the cap 259 may also include an engagement aperture46 and alignment apertures 48A, 48B to permit automated picking andplacing of the plunger into the channel.

FIG. 31 shows a reaction vessel 268 according to another embodiment ofthe invention. The vessel 268 has a plunger 270 that differs from theplunger of the preferred embodiment (described above with reference toFIGS. 1-2). The plunger 270 includes a stem 272 and an elastomeric ring274 encircling the stem 272. When the plunger 270 is inserted into thechannel 28, the ring 274 establishes a seal with the walls of thechannel. With the seal established, further insertion of the plunger 22into the channel 28 compresses the air in the channel and creates thedesired pressurization of the chamber 17 (e.g., 2 to 50 psi above theambient pressure, or more preferably 8 to 15 psi above the ambientpressure, as previously described in the preferred embodiment). Thewalls of the channel 28 may have pressure control grooves if desired, asexplained above with reference to FIGS. 7A-7D, to provide a controlledpressure stroke of the plunger 270. Alternatively, the pressure controlgrooves may be omitted so that the pressure stroke begins as soon as thering 274 enters the channel 28 and establishes a seal with the walls ofthe channel.

The plunger 270 also includes two flanges 276A, 276B extending radiallyfrom the stem 272. The flanges 276A, 276B are positioned on oppositesides of the ring 274 to hold the ring in a fixed position on the stem272. The plunger 270 may optionally have a head 278 at the end of thestem 272 for providing a physical barrier against evaporation or refluxof the reaction mixture in the chamber 17, similar to the tonguepreviously described with reference to FIG. 29. With the exception ofthe elastomeric ring 274, the plunger 270 is preferably fabricated as aone-piece polymeric part (e.g., polypropylene or polycarbonate) usingknown injection molding processes. After the body of the plunger 270 ismolded, the ring 274 is stretched over the head 278 and positioned onthe stem 272 between the flanges 276A, 276B. The ring 274 may compriseany suitable elastomeric material, e.g., a thermal plastic elastomer(TPE) or silicone. As shown in FIG. 32, the plunger cap 280 mayoptionally include an engagement aperture 46 and alignment apertures48A, 48B to permit automated picking and placing of the plunger into thechannel.

FIG. 33 shows an alternative embodiment of the invention in which thepressurization of the vessel 12 is performed by a pick-and-place machine282 having a machine head 284. The machine head 284 has an axial bore286 for communicating with the channel 28. The pick-and-place machine282 also includes a regulated pressure source in fluid communicationwith the bore 286 for pressurizing the vessel 12 through the bore 286.The pressure source may comprise, e.g., a syringe pump, compressed airsource, pneumatic pump, or connection to an external air supply.

The apparatus also preferably includes a disposable adapter 288 forplacing the bore 286 in fluid communication with the channel 28. Theadapter 288 has an axial bore 290 that connects the bore 286 in themachine head to the channel 28 in the vessel. The adapter 288 is sizedto be inserted into the channel 28 such that the adapter establishes aseal with the walls of the channel. The adapter 282 preferably comprisesan elastomeric material, e.g., a thermal plastic elastomer (tpe) orsilicone. The adapter 288 preferably includes a one-way valve 292 (e.g.,a check valve) for preventing fluid from escaping from the vessel 12.

In operation, the vessel 12 is preferably placed into a heat-exchangingmodule and filled with a reaction mixture as previously described in thepreferred embodiment. The vessel may be filled manually by a humanoperator, or alternatively, the pick-and-place machine 282 may include apipette for filling the vessel. After the chamber 17 is filled with thereaction mixture, the machine head 284 picks up the adapter 288 andinserts the adapter into the channel 28. To pick and place the adapter288, the machine head 284 preferably has a collet for gripping andreleasing the adapter 288. Alternatively, the machine head may be sizedto establish a press or friction fit with the adapter 288. When insertedinto the channel 28, the adapter 288 establishes a seal with the wallsof the channel. The pick-and-place machine 282 then transmits gas,preferably air, from the pressure source into the channel 28 to increasethe pressure in the chamber 17. The flow of air into the vessel 12 isstopped when the desired pressurization of the chamber 17 is achieved.

The desired pressurization of the chamber 17 in this embodiment is thesame as that described in the preferred embodiment above. As shown inFIG. 5, the pressure in the chamber 17 should be sufficiently high toensure that the flexible major walls 18 of the chamber outwardly expandto contact and conform to the surfaces of the plates 50A, SOB. Thepressure should not be so great, however, that the walls 18 burst,become unattached from the frame 16, or deform the frame or plates. Itis presently preferred to pressurize the chamber 17 to a pressure in therange of 2 to 50 psi above ambient pressure. This range is preferredbecause 2 psi is generally enough pressure to ensure conformity betweenthe flexible walls 18 and the surfaces of the plates 50A, SOB, whilepressures above 50 psi may cause bursting of the walls 18 or deformationof the frame 16 or plates 50A, SOB. More preferably, the chamber 17 ispressurized to a pressure in the range of 8 to 15 psi above ambientpressure. This range is more preferred because it is safely within thepractical limits described above to allow for any manufacturing oroperational deviations from specification.

Referring again to FIG. 33, the machine head 284 is disengaged from theadapter 288 following the pressurization of the vessel 12. When themachine head 284 is disengaged from the adapter 288, the valve 292prevents fluid from escaping from the vessel 12. Thus, the chamber 17remains pressurized for thermal processing and the vessel 12 iseffectively sealed to prevent the reaction mixture in the vessel fromcontaminating the external environment. The remaining operation of thisembodiment is analogous to the operation of the preferred embodimentdescribed above.

FIG. 34 shows another embodiment of the invention in which the fillingand pressurization of vessel 12 is performed by a pick-and-place machine300 having a machine head 302 for manipulating a needle 306. The machinehead 302 has an axial bore 304 for communicating with the needle 306.The pick-and-place machine 300 has controllable vacuum and pressuresources in communication with the bore 304 for aspirating and dispensingfluids using the needle 306. The vacuum and pressure sources maycomprise, e.g., one or more syringe pumps, compressed air sources,pneumatic pumps, vacuum pumps, or connections to external sources ofpressure. The machine head 302 engages the needle 306 using any standardneedle fitting, such as a luer lock. The needle 306 is preferably adouble-bore needle having a first bore 308A for injecting fluid into thevessel 12 and a second bore 308B for venting gas from the vessel. Forreasons that will soon be apparent, the first bore 308A has a lengthgreater than the second bore 308B.

The apparatus also includes an elastomeric plug 310 that is insertedinto the channel 28 of the vessel such that the plug forms a seal withthe walls of the channel. The needle 306 is inserted through the plug310 by the machine head 302 to fill and pressurize the chamber 17. Theelastomeric plug 310 should be self-sealing so that it seals fluidwithin the vessel 12 when the needle 306 is withdrawn from the plug 310.The plug 310 is preferably inserted into the channel 28 duringmanufacture of the vessel 12. Alternatively, the plug 310 may beinserted into the channel 28 just prior to using the vessel 12, e.g.,the plug may be inserted by a robotic arm or machine tip of thepick-and-place machine 300 or the plug may be manually inserted by ahuman operator.

In operation, the vessel 12 is preferably placed into a heat-exchangingmodule as previously described in the preferred embodiment, e.g., by thepick-and-place machine 300 or by human hands. The vessel 12 is thenfilled and pressurized by the pick-and-place machine 300 as follows. Themachine head 302 picks up the needle 306 and aspirates the reactionmixture into the needle through the first bore 308A. The machine head302 then inserts the needle through the plug 310 such that the firstbore 308A is in fluid communication with the channel 28 and such thatthe second bore 308B has one end disposed in the channel 28 and a secondend positioned outside of the vessel 12 and plug 310. The pick-and-placemachine 300 then dispenses the reaction mixture into the chamber 17through the first bore 308A of the needle. As the chamber 17 is filled,displaced air in the vessel 12 is vented to the atmosphere through thesecond bore 308B.

As shown in FIG. 35, the machine head 302 then partially retracts theneedle 306 from the plug 310 after the chamber 17 is filled with thereaction mixture. The needle 306 is partially retracted such that theend of the first bore 308A is still in fluid communication with thechannel 28, but the end of the second bore 308B is enclosed within theplug 310. In this position, the second bore 308B can no longer vent airfrom the channel 28. The pick-and-place machine 300 then flows gas,preferably air, from the controllable pressure source into the channel28 through the first bore 308A to increase pressure in the chamber 17.The machine 282 then stops the flow of air when the desiredpressurization of the chamber 17 is achieved.

The desired pressurization of the chamber 17 in this embodiment is thesame as that described in previous embodiments, e.g., 5 to 50 psi andmore preferably 8 to 15 psi for the reasons discussed above. Followingpressurization, the machine head 302 fully retracts the needle 306 fromthe plug 310, and the plug 310 self seals to maintain the desiredpressure in the vessel 12 for thermal processing. The needle 306 ispreferably disposable to prevent cross contamination of fluid samples.The remaining operation of this embodiment is analogous to the operationof the preferred embodiment described above.

FIG. 36 shows a slightly different embodiment of the invention in whichthe machine head 302 manipulates a single-bore needle 312 to fill andpressurize the chamber 17 in a single step. In operation, the machinehead 302 picks up the needle 312 and aspirates the reaction mixture intothe needle. The machine head 302 then inserts the needle 312 through theplug 310 and dispenses the reaction mixture into the chamber 17. It ispresently preferred that the chamber 17 be filled from the bottom up byinitially inserting the needle 312 close to the bottom of the chamber 17and by slowly retracting the needle as the chamber 17 is filled. Fillingthe chamber 17 in this manner reduces the likelihood that air bubbleswill form in the chamber.

Referring to FIG. 37, as the reaction mixture is added to the chamber17, the mixture displaces air in the vessel. The displaced air istrapped between the liquid surface level S and the plug 310 so that theair compresses in the channel 28. The compression of the air is usuallysufficient to cause the desired pressurization of the chamber 17, e.g.,2 to 50 psi above the ambient pressure, and more preferably 8 to 15 psiabove the ambient pressure.

Thus, the filling of the chamber 17 also provides for quick andconvenient pressurization in a single step. Alternatively, thepick-and-place machine 300 may be programmed to increase or decrease thepressure in the vessel 12 by adding air to the channel 28 or releasingair from the channel through the needle 312, as appropriate, to achievethe desired pressure in the chamber 17. The machine 300 preferablyincludes a pressure regulator for this purpose. Suitable pressureregulators are well known in the art.

After the desired pressurization of the chamber 17 is achieved, themachine head 302 retracts the needle 312 from the plug 310, and the plug310 seals itself to maintain the pressure in the vessel 12 for thermalprocessing. Many variations to this embodiment are possible. Forexample, there may be low pressure or a vacuum in the vessel 12 prior toadding the reaction mixture to the chamber 17. To fill and pressurizethe chamber 17, the pick-and-place machine 300 first dispenses thereaction mixture into the chamber 17 through the needle 312 and retractsthe end of the needle into the channel 28. The machine 300 then flowsair from the controllable pressure source into the channel 28 throughthe needle 312 to achieve the desired pressurization of the chamber 17.The machine head 302 then retracts the needle 312 from the plug 310, andthe plug 310 seals itself to maintain the pressure in the vessel 12 forthermal processing. The remaining operation of this embodiment is thesame as the operation of the preferred embodiment described above.

FIG. 38 shows another embodiment of the invention in which the sealingand pressurization of vessel 12 is performed by a press 314 having aheated platen 316 for heat sealing a film or foil 318 to the portion ofthe frame 16 forming the port 14. The foil 18 is preferably a laminatecomprising a layer of metal (e.g., aluminum) on top of a layer ofpolymeric material (e.g., polypropylene or polyester). In operation, thevessel 12 is preferably placed in a holder (e.g., a tray or nest) thatmoves on an assembly line for automated filling, sealing, andpressurization of the vessel. In a first step, the chamber 17 of thevessel is filled with a reaction mixture using, e.g., a pipette orsyringe. After the chamber 17 is filled, the foil 318 is placed on topof the port 14 with the metal layer facing up. The foil 318 may beplaced on the vessel manually by a human operator, or more preferably,by the robotic arm of a pick-and-place machine. The vessel 12 is thenmoved under the heated platen 316 for sealing and pressurization. Theplaten 316 is then pressed to the top of the vessel 12 and the platen316 heat seals the foil 318 to the vessel to seal the port 14.

As shown in FIG. 39, the heat from the platen 316 also melts the topportion of the frame 16, thereby collapsing an end of the channel 28 toproduce a collapsed zone 319. The volume of the channel 28 is thereforereduced. The reduction of the volume of the channel 28 after the port issealed compresses air trapped in the channel and causes the desiredpressurization of the chamber 17. The desired pressurization of thechamber 17 in this embodiment is the same as that described in theprevious embodiments, e.g., 2 to 50 psi above the ambient pressure, andmore preferably 8 to 15 psi above the ambient pressure. After the vessel12 is sealed and pressurized in this manner, it is picked and placedinto one of the heat-exchanging modules 60 (FIG. 20) for thermalprocessing and optical detection. The remaining operation of thisembodiment is the same as the operation of the preferred embodimentdescribed above.

The desired pressurization of the chamber 17 may be achieved by use ofthe equation:P _(i) *V _(i) =P _(f) *V _(f);where:P_(i) is equal to the initial pressure in the vessel 12 prior to sealingthe port;V_(i) is equal to the initial volume of the channel 28 prior tocollapsing an end of the channel;P_(f) is equal to the desired final pressure in the chamber 17; andV_(f) is equal to the final volume of the channel 28 after collapsing anend of the channel.

To ensure the desired final pressure P_(f) in the chamber 17, theheat-sealing of the vessel should reduce the volume of the channel 28such that the ratio of the volumes V_(i):V_(f) is substantially equal tothe ratio of the pressures P_(f):P_(i). An engineer having ordinaryskill in the art will be able to select suitable values for the volumesV_(i) and V_(f) using the description and equation given above. Forexample, if the initial pressure P_(i) in the vessel is equal tostandard atmospheric pressure of about 14 psi, the desired finalpressure P_(f) is equal to 26 psi (the desired 12 psi above ambientpressure), and the initial volume V_(i) of the channel is equal to 150μl, then the heat sealing of the vessel should reduce the volume of thechannel to a final volume V_(f) of about 80 μl. This is just one exampleof suitable values for the initial and final volumes, and it is to beunderstood that the scope of the invention is not limited to thisexample. Many other suitable values may be selected to achieve thedesired ratios, as will be apparent to one having ordinary skill in theart.

The various embodiments of the apparatus of the present invention mayfind use in many applications. The apparatus may be utilized to performchemical reactions on samples, e.g., nucleic acid amplification, and tooptically detect amplified target sequences. Although amplification byPCR has been described herein, it will be appreciated by persons skilledin the art that the apparatus may be utilized for a variety of otherpolynucleotide amplification reactions and ligand-binding assays. Suchadditional reactions may be thermally cycled or they may be carried outat a single temperature, e.g., isothermal nucleic acid amplification.Polynucleotide amplification reactions that may be practiced in thesystem of the invention include, but are not limited to: (1) targetpolynucleotide amplification methods such as self-sustained sequencereplication (3 SR) and strand-displacement amplification (SDA): (2)methods based on amplification of a signal attached to the targetpolynucleotide, such as “branched chain” DNA amplification; (3) methodsbased on amplification of probe DNA, such as ligase chain reaction (LCR)and QB replicase amplification (QBR); (4) transcription-based methods,such as ligation activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA); and (5) various otheramplification methods, such as repair chain reaction (RCR) and cyclingprobe reaction (CPR). Other applications of the apparatus are intendedto be within the scope of the invention where those applications requirethe transfer of thermal energy to a reaction mixture and/or opticaldetection of reaction products.

SUMMARY, RAMIFICATIONS, AND SCOPE

Although the above description contains many specificities, these shouldnot be construed as limitations on the scope of the invention, butmerely as examples of some of the presently preferred embodiments. Manymodifications or substitutions may be made to the apparatus and methodsdescribed without departing from the scope of the invention. Forexample, in one alternative embodiment, the reaction vessel has only oneflexible sheet forming a major wall of the reaction chamber. The rigidframe defines the other major wall of the chamber, as well as the sidewalls of the chamber. In this embodiment, the major wall formed by theframe should have a minimum thickness of about 0.05 inches (thepractical minimum thickness for injection molding), while the flexiblesheet may be as thin as 0.0005 inches. The advantage to this embodimentis that the manufacturing of the reaction vessel is simplified, andhence less expensive, since only one flexible sheet need be attached tothe frame. The disadvantage is that the heating and cooling rates of thereaction mixture are likely to be slower since the major wall formed bythe frame will probably not permit as high a rate of heat transfer asthe thin, flexible sheet.

In addition, the apparatus only requires one thermal surface forcontacting a flexible wall of the reaction vessel and one thermalelement for heating and/or cooling the thermal surface. The advantage tousing one thermal surface and one thermal element is that the apparatusmay be manufactured less expensively. The disadvantage is that theheating and cooling rates are likely to be about twice as slow. Further,although it is presently preferred that the thermal surfaces be formedby thermally conductive plates, each thermal surface may be provided byany rigid structure having a contact area for contacting a wall of thevessel. The thermal surface preferably comprises a material having ahigh thermal conductivity, such as ceramic or metal. Moreover, thethermal surface may comprise the surface of the thermal element itself.For example, the thermal surface may be the surface of an ultrasonictransducer that contacts the flexible wall of the chamber for ultrasonicheating and/or lysing of the sample in the chamber. Alternatively, thethermal surface may be the surface of a thermoelectric device thatcontacts the wall to heat and/or cool the chamber.

The filters used in the optics assemblies may be designed to provideexcitation and emission light in any wavelength ranges of interest, notjust the specific wavelength ranges described above. The choice offluorescent dyes for any given application depends upon the analytes ofinterest. One skilled in the art will realize that differentcombinations of light sources, filters, or filter wavelengths may beused to accommodate the different peak excitation and emission spectraof the selected dyes. Moreover, although blue and green light sourcesare presently preferred, different color light sources, such asblue-green, red, or amber LEDs, may be used in the apparatus. Further,infrared or ultraviolet light sources may be used.

Moreover, although fluorescence excitation and emission detection is apreferred embodiment, optical detection methods such as those used indirect absorption and/or transmission with on-axis geometries may alsobe applied to the apparatus of the present invention. Alternativegeometries, such as on-axis alignments of light sources and detectors,can be used to monitor changes in dye concentrations and physicalconditions (temperature, pH, etc.) of a reaction by measuring absorptionof the illumination. The optics may also be used to measure time decayfluorescence. Additionally, the optics are not limited to detectionbased upon fluorescent labels. The optics system may be applicable todetection based upon phosphorescent labels, chemiluminescent labels, orelectrochemiluminescent labels.

Therefore, the scope of the invention should be determined by thefollowing claims and their legal equivalents.

1. An apparatus for controlling the temperature of a reaction mixture,the apparatus comprising: a) a reaction vessel having a chamber forholding the mixture, the vessel comprising: i) a rigid frame definingside walls of the chamber, wherein the frame further includes a port anda channel connecting the port to the chamber; and ii) at least oneflexible sheet attached to the rigid frame to form a major wall of thechamber; b) at least one thermal surface for contacting the major wall;c) an automated machine, comprising a pick-and-place machine forinserting a plunger into the channel to compress gas in the vessel,thereby increasing the pressure in the chamber, wherein the pressureincrease in the chamber is sufficient to force the major wall to conformto the thermal surface; and d) at least one thermal element for heatingor cooling the surface to induce a temperature change within thechamber.
 2. The apparatus of claim 1, wherein the vessel includes firstand second flexible sheets attached to opposite sides of the rigid frameto form opposing major walls of the chamber, the apparatus includesfirst and second thermal surfaces formed by opposing plates positionedto receive the chamber between them, and the pressure increase in thechamber is sufficient to force the major walls to contact and conform tothe inner surfaces of the plates.
 3. The apparatus of claim 2, whereineach of the plates comprises a ceramic material, and wherein each of theplates has a thickness less than or equal to 1 mm.
 4. The apparatus ofclaim 2, wherein each of the plates has a resistive heating elementcoupled thereto.
 5. The apparatus of claim 4, wherein the heatingelement comprises a film.
 6. The apparatus of claim 2, wherein each ofthe plates has a thermal mass less than 5 J/° C.
 7. The apparatus ofclaim 2, wherein each of the plates has a thermal mass less than 3 J/°C.
 8. The apparatus of claim 2, wherein each of the plates has a thermalmass less than 1 J/° C.
 9. The apparatus of claim 2, further comprisinga support structure for holding the plates in an opposing relationshipto each other, the support structure comprising: a) a mounting platehaving a slot therein; b) spacing posts extending from the mountingplate on opposite sides of the slot, wherein each of the spacing postshas indentations formed on opposite sides thereof for receiving theedges of the plates; and c) retention clips for holding the edges of theplates in the indentations.
 10. The apparatus of claim 1, wherein theframe includes an inner surface defining the channel, and wherein theinner surface has at least one pressure control groove formed therein,the pressure control groove extending to a predetermined depth in thechannel to allow gas to escape from the vessel until the plunger reachesthe predetermined depth.
 11. The apparatus of claim 1, wherein theplunger has a pressure stroke sufficient to increase the pressure in thechamber to at least 2 psi above the ambient pressure external to thevessel.
 12. The apparatus of claim 1, wherein the automated machinecomprises: a) a machine head having an axial bore for communicating withthe channel of the vessel; and b) a pressure source for pressurizing thechamber through the bore in the machine head.
 13. The apparatus of claim12, further comprising an adapter for placing the bore in fluidcommunication with the channel, wherein the adapter is sized to beinserted into the channel such that the adapter establishes a seal withthe walls of the channel.
 14. The apparatus of claim 13, wherein theadapter includes a valve for preventing fluid from escaping from thevessel.
 15. The apparatus of claim 1, further comprising an elastomericplug inserted into the channel, wherein the automated machine comprises:a) means for inserting a needle through the plug; and b) a pressuresource for injecting fluid into the vessel through the needle.
 16. Theapparatus of claim 15, wherein the needle includes a first bore fordispensing the fluid into the vessel and a second bore for venting gasfrom the vessel, and wherein the first bore has a length greater thanthe second bore.
 17. The apparatus of claim 1, wherein the automatedmachine comprises a platen for heat sealing a film or foil to the vesselto seal the port and reduce the volume of the channel.
 18. The apparatusof claim 1, wherein: a) at least two of the side walls of the chamberare optically transmissive and angularly offset from each other; b) theapparatus further comprises an optics system having at least one lightsource for exciting the mixture through a first one of the opticallytransmissive side walls and having at least one detector for detectinglight emitted from the chamber through a second one of the opticallytransmissive side walls.
 19. The apparatus of claim 18, wherein: a) theapparatus includes first and second thermal surfaces formed by opposingplates positioned to receive the chamber of the vessel between them; andb) each of the plates has first and second edges angularly offset fromeach other by substantially the same angle that the opticallytransmissive side walls are offset from each other, and the plates arepositioned to receive the chamber between them such that the firstoptically transmissive side wall is positioned substantially adjacentand parallel to the first bottom edge of each plate and such that thesecond optically transmissive side wall is positioned substantiallyadjacent and parallel to the second bottom edge of each plate.
 20. Theapparatus of claim 18, wherein the optically transmissive side walls areangularly offset from each other by about 90°.
 21. The apparatus ofclaim 18, wherein at least two additional side walls of the chamber haveretro-reflective faces.
 22. The apparatus of claim 18, wherein the ratioof the width the chamber to the thickness of the chamber is at least4:1, and wherein the chamber has a thickness in the range of 0.5 to 2mm.
 23. The apparatus of claim 18, wherein the plates, thermal element,and optics system are incorporated into a heat-exchanging module, theapparatus further comprises a base instrument for receiving theheat-exchanging module, and the base instrument includes processingelectronics for controlling the operation of the module.
 24. Theapparatus of claim 23, wherein the heat-exchanging module furthercomprises a housing and a cooling element disposed within the housingfor cooling the reaction mixture contained in the chamber.
 25. Theapparatus of claim 23, wherein the base instrument is constructed toreceive and control a plurality of such heat-exchanging modules.
 26. Theapparatus of claim 25, further comprising at least one computer forcontrolling the base instrument.
 27. An apparatus for controlling thetemperature of a reaction mixture, the apparatus comprising: a) areaction vessel having a reaction chamber and at least one port foradding fluid to the chamber, and wherein the chamber has at least oneflexible wall; b) a thermal surface for contacting the flexible wall; c)an automated machine for increasing the pressure in the chamber,comprising i) a machine head having a bore for communicating with thevessel; and ii) a pressure source for pressuring the chamber through themachine head, wherein the pressure increase in the chamber is sufficientto force the flexible wall to contact and conform to the thermalsurface; and d) at least one thermal element for heating or cooling thethermal surface to induce a temperature change within the chamber. 28.The apparatus of claim 27, wherein the vessel further comprises a firstand a second major flexible wall, and wherein the apparatus includesfirst and second thermal surfaces formed by opposing plates positionedto receive the chamber of the vessel between them, and wherein each ofthe plates has a heating element coupled thereto.
 29. The apparatus ofclaim 28, wherein each of the plates has a thermal mass less than 5 J/°C.
 30. The apparatus of claim 28, wherein each of the plates has athermal mass less than 1 J/° C.
 31. The apparatus of claim 27, whereinthe vessel includes a channel connecting the port to the chamber, andwherein the automated machine comprises a pick-and-place machine forinserting a plunger into the channel to compress gas in the vessel. 32.The apparatus of claim 27, further comprising an adapter for placing themachine head in fluid communication with the vessel, wherein the vesselincludes a channel connecting the port to the chamber, and wherein theadapter is sized to be inserted into the channel such that the adapterestablishes a seal with the walls of the channel.
 33. The apparatus ofclaim 27, further comprising an adapter for placing the machine head influid communication with the vessel, wherein the adapter includes avalve for preventing fluid from escaping from the vessel.
 34. Theapparatus of claim 27, wherein the automated machine further comprisesmeans for dispensing fluid into the vessel through the machine head. 35.The apparatus of claim 27, wherein the vessel further comprises: a) achannel connecting the port to the chamber; b) an elastomeric pluginserted into the channel; and wherein the automated machine furthercomprises: a) a needle for inserting through the plug; b) means forinserting the needle through the plug; and c) means for injecting fluidinto the vessel through the needle.
 36. The apparatus of claim 35,wherein the needle includes a first bore for dispensing the fluid intothe vessel and a second bore for venting gas from the vessel, andwherein the first bore has a length greater than the second bore. 37.The apparatus of claim 27, wherein the automated machine comprises aplaten for heat sealing a film or foil to the vessel to seal the port.38. The apparatus of claim 27, further comprising an optics system foroptically interrogating the mixture contained in the chamber throughfirst and second optically transmissive walls of the vessel, the opticssystem having at least one light source for exciting the mixture throughthe first wall and having at least one detector for detecting lightemitted from the chamber through the second wall.
 39. The apparatus ofclaim 38, wherein the plates, heating elements, and optics system areincorporated into a heat-exchanging module, the apparatus furthercomprises a base instrument for receiving the heat-exchanging module,and the base instrument includes processing electronics for controllingthe operation of the module.
 40. The apparatus of claim 28, wherein eachof the plates comprises a ceramic material, and wherein each of theplates has a thickness less than or equal to 1 mm.
 41. The apparatus ofclaim 28, wherein the heating element comprises a film.
 42. Theapparatus of claim 28, further comprising a support structure forholding the plates in an opposing relationship to each other, thesupport structure comprising: a) a mounting plate having a slot therein;b) spacing posts extending from the mounting plate on opposite sides ofthe slot, wherein each of the spacing posts has indentations formed onopposite sides thereof for receiving the edges of the plates; and c)retention clips for holding the edges of the plates in the indentations.43. The apparatus of claim 31, wherein an inner surface of the channelhas at least one pressure control groove formed therein, the pressurecontrol groove extending to a predetermined depth in the channel toallow gas to escape from the vessel until the plunger reaches thepredetermined depth.
 44. The apparatus of claim 43, wherein the plungerhas a pressure stroke sufficient to increase the pressure in the chamberto at least 2 psi above the ambient pressure external to the vessel. 45.The apparatus of claim 27, wherein a rigid frame defines side walls ofthe chamber, and wherein: a) at least two of the side walls of thechamber are optically transmissive and angularly offset from each other;b) the apparatus further comprises an optics system having at least onelight source for exciting the mixture through a first one of theoptically transmissive side walls and having at least one detector fordetecting light emitted from the chamber through a second one of theoptically transmissive side walls.
 46. The apparatus of claim 45,wherein: a) the apparatus includes first and second thermal surfacesformed by opposing plates positioned to receive the chamber of thevessel between them; and b) each of the plates has first and secondedges angularly offset from each other by substantially the same anglethat the optically transmissive side walls are offset from each other,and the plates are positioned to receive the chamber between them suchthat the first optically transmissive side wall is positionedsubstantially adjacent and parallel to the first bottom edge of eachplate and such that the second optically transmissive side wall ispositioned substantially adjacent and parallel to the second bottom edgeof each plate.
 47. The apparatus of claim 45, wherein the opticallytransmissive side walls are angularly offset from each other by about90°.
 48. The apparatus of claim 45, wherein at least two additional sidewalls of the chamber have retro-reflective faces.
 49. The apparatus ofclaim 45, wherein the ratio of the width of the chamber to the thicknessof the chamber is at least 4:1, and wherein the chamber has a thicknessin the range of 0.5 to 2.0 mm.
 50. The apparatus of claim 39, whereinthe heat-exchanging module further comprises a housing and a coolingelement disposed within the housing for cooling the reaction mixturecontained in the chamber.
 51. The apparatus of claim 39, wherein thebase instrument is constructed to receive and control a plurality ofsuch heat-exchanging modules.
 52. The apparatus of claim 51, furthercomprising at least one computer for controlling the base instrument.